Importation of mitochondrial protein by an enhanced allotopic approach

ABSTRACT

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3′UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3′UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

FIELD OF THE INVENTION

The present invention relates to the field of cell biology, moleculargenetics, and medicine. It more particularly relates to the importationof proteins into the mitochondrion of animal and human cells.

BACKGROUND OF THE INVENTION

Mitochondria occupy a central position in the overall metabolism ofeukaryotic cells; hence the oxidative phosphorylation (OXPHOS), theKrebs's cycle, the urea cycle, the heme biosynthesis and the fatty acidoxidation take place within the organelle. Recently, another major rolefor mitochondria in determining the cellular life span was established,as they are recognized to be a major early mediator in the apoptoticcascade. Mitochondria are also a major producer of reactive oxygenspecies (ROS) causing oxidative stress and therefore inducers of celldeath.

Primary defects in mitochondrial function are implicated in over 120diseases and the list continues to grow, they encompass an extraordinaryassemblage of clinical problems, commonly involving tissues that havehigh energy requirements, such as retina, heart, muscle, kidney,pancreas and liver. Their incidence is estimated of 1 in 5,000 livebirths. Indeed, combining epidemiological data on childhood and adultmitochondrial diseases suggests this prevalence as minimum, and could bemuch higher. Therefore, mitochondrial pathologies are considered amongthe most common genetically determined diseases, and are a major healthissue since they remain inaccessible to both curative and palliativetherapies.

Mitochondrion is assembled with proteins encoded by genes distributedbetween mitochondrial and nuclear genomes. These genes include thoseencoding the structural proteins of the respiratory chain complexes I-V,their associated substrates and products, the proteins necessary formitochondrial biogenesis, the apparatus to import cytoplasmicallysynthesized precursors and the proteins necessary for mitochondrialassembly and turnover. Studies leading to the identification of genesinvolved in mitochondrial disorders have made considerable progress inthe last decade. Indeed, numerous mutations in both mitochondrial DNAand a number of nuclear genes have been reported in association with astriking diversity of clinical presentations.

Approximately half of human mitochondrial disorders are caused bypathogenic point mutations of mtDNA, one-third of which are located incoding genes. There is currently no treatment for any of thesedisorders, a possible therapeutic approach is to introduce in thenucleus a wild-type copy of the gene mutated in the mitochondrial genomeand import normal copies of the gene product into mitochondria from thecytosol. This approach has been termed “allotopic expression”.

There have already some reports describing that engineerednucleus-localized version of some mtDNA genes could be expressed inmammalian cells. For example, in a Leigh's disease case, a plasmid wasconstructed in which the mitochondrial targeting signal of the nuclearencoded COX8 gene was appended to a recoded mitochondrial ATP6 gene,mutated in patients. Stably transfected cells from patients present animprovement of growth in galactose medium and a mild increase in ATPsynthesis, however the amount of Atp6 protein imported into mitochondriawas relatively low (18.5%), implying that the precursor was not importedefficiently (Manfredi, G., et al., Rescue of a deficiency in ATPsynthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene to thenucleus. Nature Genet., 2002. 30: p. 394-399).

Oco-Cassio and co-workers have demonstrated that allotopic expression ofapocytochrome b and ND4 into Cos-7 and HeLa cells, did not lead to anefficient mitochondrial import of these proteins (Oca-Cossio, J., etal., Limitations of allotopic expression of mitochondrial genes inmammalian cells. Genetics, 2003. 165: p. 707-720).

Hence, up today important limitations are found to the allotopicexpression as a therapeutic approach and require optimization toovercome the significant hurdles before it can be applied in genetictherapy.

One hypothesis that can explain the poor import ability of themitochondrial protein is its high hydrophobicity. Thus, the precursorsynthesized in the cytoplasm remains stuck on the outer mitochondrialmembrane.

Mitochondria assembly depends on balanced synthesis of 13 proteinsencoded by mtDNA with more than a thousand others encoded by nuclearDNA. As the vast majority of mitochondrial polypeptides are synthesizedin the cytoplasm, there is the requirement for an efficient and specificprotein targeting system. This process involves the transport of mRNAsfrom the nucleus to the surface of mitochondria.

The inventors examined the possibility that allotopic expression of DNAsuch as mtDNA could be optimized by a targeted localization of the mRNAto the mitochondrial surface.

SUMMARY OF THE INVENTION

Mitochondrial proteins are encoded by nucleic acids which are located inthe mitochondrion, i.e. mitochondrial nucleic acids (mtDNA, mtRNA), aswell as by nucleic acids which originate from the nucleus, i.e. nuclearnucleic acids (nDNA, nRNA).

The inventors describe an enhanced allotopic approach for importation ofproteins into the mitochondrion. The present invention provides means,including compositions and methods, which enable mitochondrialimportation at enhanced efficiency and stability compared to prior arttechniques. The means of the invention enable a targeted localization ofthe mRNA to the mitochondrial surface.

Compared to prior art techniques, the means of the invention enable theefficient and stable importation of protein into the mitochondrion of ananimal of human in need thereof, such as an animal or human having acellular dysfunction caused by one or several mutations in a geneencoding a mitochondrial protein.

The inventors demonstrate that mRNA sorting to the mitochondrial surfaceis an efficient way to proceed to such an allotopic expression, and thatthis mRNA sorting can be controlled by selecting appropriatemitochondrion-targeting sequence (MTS) and appropriate 3′UTR sequences.The CDS sequence which codes for the protein to be delivered into themitochondrion is guided by these appropriate MTS and 3′UTR sequencesfrom the nuclear compartment to the mitochondrion-bound polysomes (wherethe CDS is translated), and aids in an efficient translocation of amature functional protein into the mitochondria.

The inventors demonstrate that, to obtain a stabletherapeutically-effective importation, both an appropriate MTS and anappropriate 3′UTR should preferably be used.

Appropriate MTS and 3′UTR sequences correspond to those ofnuclearly-transcribed mitochondrially-targeted mRNAs. If a vector isused, it is preferred that it does not contain any 3′UTR which wouldcorrespond to the 3′UTR of a nuclearly-transcribed butnot-mitochondrially-targeted mRNAs. To the best of the inventors'knowledge, all commercially-available vectors contain such anot-mitochondrially-targeted mRNA; it is then preferred to delete thisinappropriate 3′UTR from the vector before use as mitochondrialimporter.

The means of the invention are especially adapted to animal and humancells, and more particularly to mammalian cells. They give access totherapeutically-effective means for such cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Map and sequence of COX10 MTS-nATP6, SOD2MTS-nATP6,COX10 MTS-nATP6-COX10 3′UTR, SOD2MTS-nATP6-SOD2 3′UTR obtained in thepCMV-Tag 4A vector.

FIG. 1A. The four constructs are schematically represented.

FIG. 1B. The COX10 MTS-nATP6 and SOD2MTS-nATP6 are introduced at theEcoRI restriction site of the pCMV-tag4A vector. EcoRI restriction sitesare framed. The ATG of SOD2 MTS, COX10 MTS and nATP6, are bold andunderlined. The COX10 3′UTR and the SOD2 3′UTR are inserted at the PvuIand MluI restriction sites, represented in bold. FLAG tag epitope is initalics.

FIG. 2: RT-PCR analyses of RNAs purified from HeLa transfected cells

100 ng of total RNAs were reversed transcribed and subjected to 30cycles of PCR amplification, 10% of amplified product were subjected toagarose electrophoresis.

-   -   1: Transiently transfected cells with the COX10 MTS-nATP6 vector        (SV40 3′UTR).    -   2: Transiently transfected cells with COX10 MTS-nATP6-COX10        3′UTR vector.    -   In 3 and 4 we examined RNAs from the same transfection        experiment but in this case it represents the stably transfected        cells.    -   5: HeLa transfected cells with the empty pCMV-tag4A vector.    -   6: Hela cells.

Specific oligonucleotide primers were used to detect hybrid ATP6 mRNA intransfected cells, for the MTS COX10-ATP6 product MTS COX10 and ATP6 ORF3′ were used (cf. Table 2 below, in example 1). For the amplification ofthe complete ATP6 ORF and the entire COX10 3′UTR the ATP6 ORF 5′ primerand the 3′ UTR COX10 3′ Primer were used (cf. Table 2 below, in example1). As internal control, the steady-state levels of COX6c mRNA wereexamined in all the RNA preparations using both COX6 primers shown insaid Table 2.

FIG. 3: Subcellular localization of the recoded Atp6 protein in HeLacells

Stably transfected cells with either COX10 MTS-nATP6 (SV40 3′UTR) orCOX10 MTS-nATP6-COX10 3′UTR vector were visualized by indirectimmunofluorescence using antibodies to Flag and ATP synthase subunitbeta. The punctuate pattern of Flag antibody staining indicates that thefusion Atp6 protein is efficiently transported to mitochondria in vivo,since the same pattern of mitochondria labeling was observed with thebeta subunit of ATP synthase.

FIGS. 4A and 4B: nATP6 proteins are efficiently imported intomitochondria in vivo.

FIG. 4A: Proteins were extracted from HeLa cells and HeLa transfectedcells (COX10 MTS-nATP6-SV40 3′UTR or COX10 MTS-nATP6-COX10 3′UTRvectors) and assayed for import in the absence and presence ofproteinase K (PK). Proteins were treated with 200 μg/ml of proteinase Kat 0° C. for 30 min. Then, they were separated on 4-12% polyacrylamideSDS gel and transferred into a nitrocellulose membrane. The resultingblot was probed with mouse monoclonal anti-ATP synthase subunit alpha ormouse monoclonal anti-Flag M2 antibodies.

FIG. 4B: Histograms of the amount of COX10 MTS-nATP6-Flag and Atpsynthase subunit alpha with or without proteinase K. Signals fromimmunoblots were scanned and quantified by the MultiAnalyst System(Bio-Rad). The amount of the mature ATP6 protein insensitive toproteinase K proteolysis is approximately 185% higher in cellstransfected with COX10 MTS-nATP6-COX10 3′UTR vector compared to cellsexpressing the COX10 MTS-nATP6 without COX10 3′UTR but with the SV40Poly A signal. Besides, the amount of the mature form of the recodedATP6 protein inside mitochondria is very similar to the one measured forthe naturally imported ATP synthase subunit alpha, confirming thatrecoded ATP6 proteins are efficiently translocated into the organelle.

FIGS. 5A and 5B: Map and sequence of COX10 MTS-nND1, COX10MTS-nND4,COX10 MTS-nND1-COX10 3′UTR, COX10MTS-nND4-COX10 3′UTR obtained in thepCMV-Tag 4A vector.

FIG. 5A. The four constructs are schematically represented.

FIG. 5B. The COX10 MTS-nND1 and COX10MTS-nND4 are introduced at theXhoI/SalI restriction sites of the pCMV-tag4A vector. XhoI and SalIrestriction sites are framed. The ATG of COX10 MTS, nND1 and nND4, arebold and underlined. The COX10 3′UTR is inserted at the PvuI and MluIrestriction sites, represented in bold. FLAG tag is in italics.

FIG. 6: Immunocytochemistry of G3460A LHON fibroblats

The fusion protein was visualized by indirect immunofluorescence usingantibodies to Flag. Indicative of mitochondrial import, cellstransfected with either COX10 MTS-nND1-SV40 3′UTR or COX10MTS-nND1-COX10 3′UTR vectors exhibited a typically punctuate stainingpattern, also observed with the beta subunit of ATP synthase, whichlocalize in vivo to the inner mitochondrial membrane. In contrast, cellstransfected with the empty pCMV-Tag 4A vector exhibited a very lowintensity and diffuse cytoplasmic staining when antibodies to Flag wereused.

FIG. 7: Immunocytochemistry of G11778A LHON fibroblats

The fusion protein was visualized by indirect immunofluorescence usingantibodies to Flag. Cells transfected with either COX10 MTS-nND4-SV403′UTR or COX10 MTS-nND4-COX10 3′UTR vectors exhibited a typicallypunctate staining pattern, also observed with the beta subunit of ATPsynthase, which localize in vivo to the inner mitochondrial membrane.This data indicates that ND4 is efficiently imported into mitochondria.In contrast, cells transfected with the empty pCMV-Tag 4A vectorexhibited a very low intensity and diffuse cytoplasmic staining whenantibodies to Flag were used.

FIG. 8: Growth in glucose-free medium of non-transfected fibroblastswith the G3460A mutation and transfected fibroblasts with the MTSCOX10-nND1-COX10 3′UTR vector Fibroblasts from LHON patients presentingthe G3460A mutation were stably transfected with the MTSCOX10-nND1-COX10 3′UTR vector and examined for their ability to growthon DMEM medium supplemented with 10 mM galactose. Non-transfectedfibroblasts (LHON G3460A ND1) show a severe growth defect on galactosemedium, the ability to grow on galactose was significantly improved whenthe recoded nND1 protein is expressed in stably transfected cells (LHONG3460A ND1 MTSCOX10-nND1). Cells were photographed after 6 day culture.

FIG. 9: recoded CDS of mtDNA (SEQ ID NO: 27-29)

FIG. 9 shows the human nucleic acid coding sequence of the mitochondrialATP6, ND1, ND4, recoded according to the universal genetic code (nATP6,nND1, nND4 of SEQ ID NO:27, 28 and 29, respectively). The recoded ND1and ND4 which are shown in FIG. 9 also take into account the preferredhuman codon usage (see example 2 below).

FIG. 10: illustrative human co-translational MTS and 3′UTR (SEQ ID NOS:30, 47, 31, 60)

FIG. 10 shows the sequence of human COX10 MTS (SEQ ID NO: 30), humanCOX10 3′UTR (SEQ ID NO: 47), human SOD2 MTS (SEQ ID NO: 31), and humanSOD2 3′NTR (SEQ ID NO: 60).

TABLE 5 Nucleic acid MTS Nucleic acid 3′UTR COX10 SEQ ID NO: 30 SEQ IDNO: 47 SOD2 SEQ ID NO: 31 SEQ ID NO: 60

FIGS. 11A-11I show the protein sequence coded by illustrative humanmitochondrially-targeted mRNA, as well as their respective MTS peptidesequences and their respective 3′UTR sequences. ATCC accession number isindicated for each of these protein sequences.

ACO2=Aconitase;

SOD2 Mitochondrial Superoxide dismutase;ATP5b=P synthase subunit beta;UQCRFS1=Ubiquinol-cytochrome c reductase. Rieske iron-sulfur polypeptide1;NDUFV1 NADH-ubiquinone oxidoreductase 51 kDa subunit, mitochondrialprecursor (Complex I-51KD) (CI-51KD) (NADH dehydrogenase flavoprotein1);NDUFV2=NADH dehydrogenase (ubiquinone) flavoprotein 2, 24 kDa;ALDH2=Mitochondrial aldehyde dehydrogenase 2 precursor;COX10 Heme A:farnesyltransferase;

AK2=Adenylate Kinase 2.

SEQ ID NO are as follows:

TABLE 6 MTS peptide 3′UTR Protein ACO2 SEQ ID NO: 32 SEQ ID NO: 33 SEQID NO: 48 SOD2 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 49 ATP5b SEQ IDNO: 36 SEQ ID NO: 37 SEQ ID NO: 50 UQCRFS1 SEQ ID NO: 38 SEQ ID NO: 39SEQ ID NO: 51 NDUFV1 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 52 NDUFV2SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 53 ALDH2 SEQ ID NO: 44 SEQ ID NO:45 SEQ ID NO: 54 COX10 SEQ ID NO: 46 SEQ ID NO: 59 SEQ ID NO: 55 AK2 SEQID NO: 57 SEQ ID NO: 56

FIGS. 12A, 12B, 12C: Subcellular distribution of hybrid ATP6 mRNAs inHeLa cells

A. Total RNAs extracted from cells expressing the SOD2^(MTS)ATP6-3′UTR^(SV40) (S.T 1 and S.T 2) or the SOD2^(MTS) ATP6-3′UTR^(SOD2)(S.T 3 and S.T 4) vectors were subjected to RT-PCR analysis to revealamounts of hybrid ATP6 (SOD2^(MTS) ATP6) mRNAs and endogenous SOD2, ATP6and COX6c mRNAs. The amount of RNAs used for the reverse transcription,FOR conditions and specific oligonucleotides used for each gene aresummarized in Table 9.

B. RNAs were purified from mitochondrion-bound polysomes (M-P) andfree-cytoplasmic polysomes (F-P) of stably transfected cell lines witheither SOD2^(MTS) ATP6-3′UTR^(SV40) (S.T 1 and S.T 2) or SOD2′^(MTS)ATP6-3′UTR^(SOD2) (S.T 3 and S.T 4) vectors and subjected to RT-PCRanalysis. The abundance of endogenous ATP6, SOD2 and COX6c mRNAs wasdetermined in each polysomal population using the conditions shown inTable 9.

C. Densitometric Analyses were Performed Using the Quantity One BioradSoftware System.

The difference between the amounts of hybrid ATP6 mRNAs in cellsexpressing respectively SOD2^(MTS) ATP6-3′UTR^(SV40) or SOD2^(MTS)ATP6-3′UTR^(SOD2) constructions was significant according to the pairedStudent's t-test (P<0.0034, n=6).

FIG. 13: Subcellular localization of the recoded Atp6 protein in vivo

Stably transfected cells with either the empty pCMV-tag4A vector,SOD2^(MTS) ATP6-3′UTR^(SV40) or SOD2^(MTS) ATP6-3′ UTR^(SOD2) plasmidswere visualized by indirect immunofluorescence using antibodies to Flagand ATP synthase subunit α. For each cell type visualized, a mergedimage in association with DAPI staining is shown at the right panel.Indicative of the mitochondrial localization of recoded ATP6 proteins,cells transfected with either SOD2^(MTS) ATP6-3′UTR^(SV40) or SOD2^(MTS)ATP6-3′UTR^(SOD2) plasmids showed a significant colocalization of bothFlag and ATP synthase α signals. In contrast, cells transfected with theempty vector exhibited a low diffuse cytoplasmic staining.

FIGS. 14A, 14B, 14C: Recoded ATP6 proteins are efficiently imported intomitochondria in vivo.

A. Six independent mitochondria purifications were performed with cellsstably transfected with either SOD2^(MTS) ATP6-3′UTR^(SV40) SOD2^(MTS)ATP6-3′^(SOD2) plasmids and subjected to Western blot analysis. Signalsfor the ATP6 precursors and mature forms were scanned and quantified bythe Quantity One System (Bio-Rad). No significant differences betweenthe amounts of the precursor and the mature form of the recoded ATP6proteins were observed in each cell line examined. B. Upper panel:Schematic representation of mitochondrial import intermediates. Thehydrophobic passenger protein can be trapped en route to the matrix. Inthis step, the protein can be blocked or represented an intermediate oftranslocation. This doesn't prevent the cleavage of the MTS by amitochondrial processing peptidase, the rest of the protein remainsaccessible to PK digestion and therefore if digested it becomesundetectable in the Western blot assay. The fraction of the proteincompletely translocated turns into a mature protein insensitive to PKlocated in the inner mitochondrial membrane. MM: mitochondrial matrix;OM: outer membrane; MIS: mitochondrial intermembrane space, TOM:Translocase of the Outer Membrane, TIM: Translocase of the Innermembrane.

Middle panel: Mitochondria extracted from transfected cells with eitherthe empty pCMV-Tag 4A vector, SOD2^(MTS) ATP6-3′UTR^(SV40) or SOD2^(MTS)ATP6-3′UTR^(SOD2) plasmids were subjected to Western blot essays. 20 μgof proteins were treated with 150 μg/ml of PK at 0° C. for 30 minutesand subjected to immunoblotting analysis using anti-ATP synthase subunitα and anti-Flag M2 antibodies. Densitometric analyses of experimentsperformed with six independent mitochondrial purifications wererepresented at the lower panel. We normalized values measured for thesignal of the mature form of ATP6 resistant to PK with ATPα signalrevealed after PK digestion. We then compared the value obtained forcells expressing either the SOD2^(MTS) ATP6-3′UTR^(SV40) or theSOD2^(MTS) ATP6-3′UTR3^(SOD2) plasmids. Signals from Western blots werescanned and quantified by the Quantity One System (Bio-Rad). Thedifference between the amounts of fully mitochondrial translocated ATP6protein in cells expressing respectively SOD2^(MTS) ATP6-3′UTR^(SV40) orSOD2^(MTS) ATP6-3′UTR^(SOD2) constructions was significant according tothe paired Student's t-test (P<0.0022, n=6).

C. 20 μg of mitochondria isolated from cells stably transfected withSOD2^(MTS) ATP6-3′UTR^(SOD2) vector treated with 150 μg/ml of PK and 1%Triton X100 at 0° C. for 30 min and subsequently subjected to Westernanalysis

FIG. 15: Mitochondrial import ability of ATP6 proteins based on themesohydrophobicity index

A plot developed by Claros and Vincens was used to measure mitochondrialimport ability of fusion ATP6 proteins. By this approach, the fusionSOD2^(MTS) ATP6 protein would not be importable. Mesohydrophobicity,which is the average regional hydrophobicity over a 69 amino acidregion, was calculated using Mito-ProtII. Values obtained are thefollowing: ATP6: 1.41; SOD2^(MTS) ATP6: 1.41; COX8^(MTS) ATP6: 1.41;SOD2: −1.26; COX8: −1.63.

FIG. 16: rescue of NARP cells; survival rate on galactose medium of NARPcells (mutated ATP6), and of NARP cells transfected by a SOD2 MTS-ATPvector (SOD2 MTS-ATP6-SV40 3′UTR), or by a vector of the invention (SOD2MTS-ATP6 SOD2 3′UTR); see also example 3, table 11.

FIG. 17: rescue of LHON fibroblasts; survival rate on galactose mediumof LHON fibroblasts (mutated ND1), and of LHON fibroblasts transfectedby a COX10 MTS-ND1-SV40 3′UTR vector, or by a vector of the invention(COX10 MTS-ND1-COX10 3′UTR); see also example 4, table 12.

FIG. 18: mitochondrial distribution in retinal gangion cells (RGC)transfected with the mutated version of ND1.

FIG. 19: rescue of NARP cells; anti-Flag, Mito-tracker and merged+DAPIstaining of NARP cells transfected either with SOD2 MTS ATP6-SV40 3′UTR, or with SOD2 MTS-ATP6-SOD2 3′UTR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use and control of mRNA sorting atthe surface of mitochondria.

Schematically, the present invention relates to the use of nucleic acidsequences corresponding to a co-translational MTS and of aco-translational 3′UTR, for guiding a desired mRNA (which codes adesired mitochondrial protein) from the nucleus to themitochondria-bound polysomes, and for inducing the effectivetranslocation of the translated protein into the mitochondrion.

By “co-translational”, it is herein referred to a nuclearly-encodedmitochondrially-targeted pathway.

Mitochondrion-Targeting Sequences (MTS):

Sequences known as mitochondrion-targeting signal or mitochondrialtargeting signal are referred to as MTS by the skilled person.

A MTS sequence can be identified within a protein or nucleic acidsequence by a person of ordinary skill in the art.

Most mitochondrion-targeting peptides consist of a N-terminalpre-sequence of about 15 to 100 residues, preferably of about 20 to 80residues. They are enriched in arginine, leucine, serine and alanine.Mitochondrial pre-sequences show a statistical bias of positivelycharged amino acid residues, provided mostly through arginine residues;very few sequences contain negatively charged amino acids.Mitochondrion-targeting peptides also share an ability to form anamphilic alpha-helix.

A complete description of a method to identify a MTS is available in: M.G. Claros, P. Vincens, 1996 (Eur. J. Biochem. 241, 779-786 (1996),“Computational method to predict mitochondrially imported proteins andtheir targeting sequences”), the content of which is herein incorporatedby reference.

Software is available to the skilled person to identify the MIS of agiven sequence. Illustrative software notably comprises the MitoProt®software, which is available e.g. on the web site of the Institut fürHumangenetik; Technische Universitãt München, Germany. The MitoProt®software calculates the N-terminal protein region that can support aMitochondrial Targeting Sequence and the cleavage site. Theidentification of the N-terminal mitochondrial targeting peptide that ispresent within a protein gives a direct access to the nucleic acidsequence, i.e. to the MTS (e.g. by reading the corresponding positionsin the nucleic acid sequence coding for said protein).

Illustrative human MIS peptide sequences and human 3′UTR originatingfrom human nuclearly-encoded mitochondrially-targeted mRNA are given inFIGS. 11A, 11B, 11C, 11D, 11E, 11F, 11G, 11H and 11I.

SEQ ID NOs are as follows:

TABLE 7 Illustrative human mRNAs which are nuclearly-encoded butmitochondrially-targeted MTS peptide FIG. ACO2 SEQ ID NO: 32 11A SOD2SEQ ID NO: 34 11B ATP5b SEQ ID NO: 36 11C UQCRFS1 SEQ ID NO: 48 11DNDUFV1 SEQ ID NO: 40 11E NDUFV2 SEQ ID NO: 42 11F ALDH2 SEQ ID NO: 4411G COX10 SEQ ID NO: 46 11H

3′UTR:

The 3′UTR of a RNA molecule is defined as the fragment of this RNAmolecule that extends from the STOP codon to the end of the molecule.According to the universal genetic code, there are three possible STOPcodons: TGA, TAA, TAG.

An online data base gives direct access to 3′UTR sequences.

Illustrative 3′UTR sequences which can used in accordance with theinvention are shown in FIGS. 11A, 11B, 110, 11D, 11E, 11F, 11G, 11H and11I (Accession numbers of these sequences are also indicated).

SEQ ID NOs are as follows:

TABLE 8 Illustrative human mRNAs which are nuclearly-encoded butmitochondrially-targeted 3′UTR FIG. ACO2 SEQ ID NO: 33 11A SOD2 SEQ IDNO: 35 11B ATP5b SEQ ID NO: 37 11C UQCRFS1 SEQ ID NO: 39 11D NDUFV1 SEQID NO: 41 11E NDUFV2 SEQ ID NO: 43 11F ALDH2 SEQ ID NO: 45 11G COX10 SEQID NO: 47 10 AK2 SEQ ID NO: 57 11I

Vectors of the Invention:

The present invention relates to a vector which is adapted to theefficient and stable delivery of a protein into the mitochondrion of ananimal or human cell, preferably a mammalian cell, most preferably ahuman cell.

The vector of the invention can be produced in the form of a recombinantvector. Advantageously, the vector of the invention is an expressionvector.

A vector of the invention comprises:

-   -   at least one nucleic acid sequence encoding a        mitochondrion-targeting signal (also referred to as: MTS nucleic        acid sequence),    -   at least one nucleic acid sequence which encodes said protein to        be delivered, in accordance with the universal genetic code        (also referred to as: CDS), and    -   at least one 3′ nucleic acid sequence.

Said at least one MTS nucleic acid sequence is a co-translational MTSnucleic acid sequence, or a conservative fragment or variant thereof.

Said at least one 3′ nucleic acid sequence is a co-translational 3′UTRnucleic acid sequence or the DNA sequence of such a co-translational3′UTR, or a conservative fragment or variant thereof.

Preferably, said vector does not comprise a non-co-translational 3′UTR.

Said vector does not use a post-translation importation pathway, butuses a co-translation importation pathway from nucleus to saidmitochondrion.

The delivery of protein according to the invention not only comprisesthe translocation of the protein-encoding nucleic acid from nucleustowards mitochondrion, but also comprises the translation of the encodedprotein in the cytosol but at proximity of the mitochondrion (onmitochondrion-bound polysomes), and the effective importation of thetranslated protein into said mitochondrion. The invention provides avery advantageous importation mechanism compared to prior arttechniques, which provided the mitochondrion with mature proteins at anunsatisfactory level of efficiency.

The present invention further provides a stable importation of saidprotein into the mitochondrion. It means that the protein rescueobtained by the invention is a rescue that is stable over time: thefibroblasts of a LHON patient transfected by a vector of the invention(expressing N©1) has grown in vitro for at least 20 days on a galactoseculture medium. To the best of the inventors' knowledge, this is thefirst time that a culture of LHON patient fibroblasts can be keptgrowing for such a long period on a galactose medium.

The invention thus relates to a vector adapted to the efficient andstable delivery of a protein into the mitochondrion of an animal orhuman cell, preferably a mammalian cell, most preferably a human cell,which comprises:

-   -   at least one mitochondrion-targeting nucleic acid sequence (MTS        nucleic acid sequence),    -   at least one nucleic acid sequence which encodes said protein in        accordance with the universal genetic code (CDS), and    -   at least one 3′ nucleic acid sequence, which is located in 3′ of        said at least one MTS nucleic acid sequence and said at least        one CDS.

Preferably, said at least MTS nucleic acid sequence is in 5′ positioncompared to said at least one CDS sequence, whereby the vector has thefollowing structure (from 5′ to 3′): at least one MTS nucleic acidsequence—at least one CDS—at least one 3′UTR.

Said at least one MTS nucleic acid sequence is:

-   -   the MTS RNA sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, preferably the MTS RNA sequence        of a naturally-occurring nuclearly-encoded        mitochondrially-targeted m RNA, or    -   the cDNA sequence of such a MTS RNA sequence, or    -   a DNA sequence coding for such a MTS RNA sequence in accordance        with the universal genetic code, or    -   a conservative variant or fragment of such a RNA or cDNA or DNA        MTS sequence, which derives therefrom by deletion and/or        substitution and/or addition of one or several nucleotides, but        has retained a mitochondrion-targeting function.

In other words, said at least one MTS nucleic acid sequence is:

-   -   the RNA sequence of a MTS which targets a (preferably        naturally-occurring) nuclearly-transcribed mRNA to the surface        of a mitochondrion in a cell collected from a healthy animal or        human being, or in a normal animal or human cell, or    -   the cDNA sequence of such a MTS RNA sequence, or    -   a DNA sequence coding for such a MTS RNA sequence in accordance        with the universal genetic code, or    -   a conservative variant or fragment of such a RNA or cDNA or DNA        MTS sequence, which derives therefrom by deletion and/or        substitution and/or addition of one or several nucleotides, but        has retained a mitochondrion-targeting function.

Preferably, said at least one MTS nucleic acid sequence is:

-   -   the cDNA sequence of a MTS of a nuclearly-encoded        mitochondrially-targeted mRNA, or    -   a conservative variant or fragment of such a cDNA sequence,        which derives therefrom by deletion and/or substitution and/or        addition of one or several nucleotides, but has retained a        mitochondrion-targeting function.

Said at least one 3′ nucleic acid sequence is:

-   -   the 3′UTR sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, preferably the 3′UTR sequence of        a naturally-occurring nuclearly-encoded mitochondrially-targeted        mRNA, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding for such a 3′UTR sequence in accordance        with the universal genetic code, or    -   a conservative variant or fragment of such a RNA or cDNA or DNA        3′UTR sequence, which derives therefrom by deletion and/or        substitution and/or addition of one or several nucleotides, and        which, when replacing the wild-type 3′UTR of said        nuclearly-encoded mitochondrially-targeted mRNA, still allows        for a mitochondrial targeting of the resulting mRNA.

In other words, said at least one 3′ nucleic acid sequence is:

-   -   the RNA sequence of the 3′UTR of a nuclearly-transcribed        mitochondrially-targeted mRNA, i.e. the RNA sequence of the        3′UTR of a (preferably naturally-occurring)        nuclearly-transcribed RNA which is targeted to the surface of a        mitochondrion in a cell collected from a healthy animal or human        being, or in a normal animal or human cell, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding for such a 3′UTR sequence in accordance        with the universal genetic code, or    -   a conservative variant or fragment of such a RNA or cDNA or DNA        3′UTR sequence, which derives therefrom by deletion and/or        substitution and/or addition of one or several nucleotides, and        which, when replacing the wild-type 3′UTR of said        nuclearly-encoded mitochondrially-targeted mRNA, still allows        for a mitochondrial targeting of the resulting mRNA.

Preferably, said at least one 3′ nucleic acid sequence is:

-   -   the cDNA sequence of the 3′UTR sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, or    -   a conservative variant or fragment of such a cDNA sequence,        which derives therefrom by deletion and/or substitution and/or        addition of one or several nucleotides, and which, when        replacing the wild-type 3′UTR of said nuclearly-encoded        mitochondrially-targeted mRNA, still allows for a mitochondrial        targeting of the resulting mRNA.

The resulting vector does not use a post-translation importationpathway, but uses a co-translation importation pathway from nucleus tosaid mitochondrion.

Preferably, said vector (inserted nucleic acid construct included) doesnot comprise any sequence which would be identical to:

-   -   the 3′UTR of a naturally-occurring mRNA which is a (preferably        naturally-occurring) nuclearly-transcribed but        non-mitochondrially-targeted mRNA, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding such a 3′UTR in accordance with the        universal genetic code.

Preferably, said vector (inserted nucleic acid construct included) doesnot comprise any sequence which would be identical to:

-   -   the 3′UTR of a mRNA which is not targeted to the surface of a        mitochondrion, and preferably the 3′UTR of a naturally-occurring        mRNA which is not targeted to the surface of a mitochondrion, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding such a naturally-occurring mRNA 3′UTR in        accordance with the universal genetic code.

The present invention more particularly relates to a vector adapted tothe efficient and stable delivery of a protein into the mitochondrion ofa mammalian cell, which comprises:

-   -   at least one mitochondrion-targeting nucleic acid sequence        (referred to as MTS nucleic acid sequence),    -   at least one nucleic acid sequence which encodes said protein in        accordance with the universal genetic code (referred to as CDS        sequence), and    -   at least one 3′ nucleic acid sequence, which is located in 3′ of        said at least one MTS nucleic acid sequence and of said at least        one CDS,        wherein said at least one MTS nucleic acid sequence is:    -   the cDNA sequence of a MTS of a nuclearly-encoded        mitochondrially-targeted mRNA, or    -   a conservative variant or fragment of such a cDNA sequence,        which derives therefrom by deletion and/or substitution and/or        addition of one or several nucleotides, but has retained a        mitochondrion-targeting function,        wherein said at least one 3′ nucleic acid sequence is:    -   the cDNA sequence of the 3′UTR sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, or    -   a conservative variant or fragment of such a cDNA sequence,        which derives therefrom by deletion and/or substitution and; or        addition of one or several nucleotides, and which, when        replacing the wild-type 3′UTR of said naturally-occurring mRNA,        still allows for a mitochondrial targeting of the resulting        mRNA,        wherein said vector does not comprise any sequence which would        be identical to:    -   the 3′UTR of a naturally-occurring mRNA which is a        nuclearly-transcribed but not-mitochondrially-targeted mRNA, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding for such a naturally-occurring mRNA 3′UTR        in accordance with the universal genetic code,        whereby said vector does not use a post-translation importation        pathway, but uses a co-translation importation pathway from        nucleus to said mitochondrion.

Said at least one MTS nucleic acid sequence can e.g. be the MTS nucleicacid sequence of ACO2, or of SOD2, or of ATP5b, or of UQCRFS1, or ofNDUFV1, or of NDUFV2, or of ALDH2, or of COX10.

Said at least one MTS nucleic acid sequence may thus code for a sequenceof SEQ ID NO:32, or SEQ ID NO:34, or SEQ ID NO:36, or SEQ ID NO:38, orSEQ ID NO:40, or SEQ ID NO:42, or SEQ ID NO:44, or SEQ ID NO:46 the MTSpeptidic or polypeptidic sequence of human ACO2, SOD2, ATP5b, UQCRFS1,NDUFV1, NDUFV2, ALDH2, COX10, respectively; see FIGS. 11A-11H).

Preferably, said at least one MTS nucleic acid sequence is the MTSnucleic acid sequence of ACO2, or of SOD2, or of ATP5b, or of COX10.

Said at least one MTS nucleic acid sequence may thus code for a sequenceof SEQ ID NO:32, or SEQ ID NO:34, or SEQ ID NO:36, or SEQ ID NO:46 (=theMTS peptidic or polypeptidic sequence of human ACO2, SOD2, ATP5b, COX10,respectively).

Preferably, said at least one MTS nucleic acid sequence is SEQ ID NO:30, or SEQ ID NO: 31 (MTS nucleic acid sequence of human COX10 and SOD2,respectively; see FIG. 10).

Said at least one 3′ nucleic acid sequence can be e.g. be:

-   -   the 3′UTR sequence of ACO2, or of SOD2, or of ATP5b, or of        UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10,        or of AK2, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding for such a 3′UTR sequence in accordance        with the universal genetic code.

Said at least one 3′ nucleic acid sequence may thus comprise or consistof those of SEQ ID NO: 33, or SEQ ID NO: 35, or SEQ ID NO: 37, or SEQ IDNO: 39, or SEQ ID NO: 41, or SEQ ID NO: 43, or SEQ ID NO: 45, or SEQ IDNO: 47, or SEQ ID NO: 57 (=the sequences corresponding to the human3′UTR of ACO2, SOD2, ATP5b, UQCRFS1, NDUFV1, NDUFV2, ALDH2, COX10, AK2,respectively; see FIGS. 10 and 11A-11I).

Preferably, said at least one 3′ nucleic acid sequence is

-   -   the 3′UTR sequence of ACO2, or of SOD2, or of ATP5b, or of        COX10, or of AK2, or    -   the cDNA sequence of such a 3′UTR, or    -   a DNA sequence coding for such a 3′UTR sequence.

Said at least one 3′ nucleic acid sequence may thus comprise or consistof SEQ ID NO: 33, or SEQ ID NO: 35, or SEQ ID NO: 37, or SEQ ID NO: 47,or SEQ ID NO: 57 (=the sequences corresponding to the human 3′UTR ofACO2, SOD2, ATP5b, COX10, AK2, respectively; see FIGS. 10 and 11A-11I).

Preferably, said at least one 3′ nucleic acid sequence is SEQ ID NO: 35(human SOD2 3′UTR), or SEQ ID NO: 47 (human COX10 3′UTR).

Said at least one CDS nucleic acid sequence can be a RNA, a cDNA or aDNA sequence. Preferably, said at least one CDS sequence is a cDNAsequence.

According to a very advantageous aspect of the invention, said at leastone CDS may be any nucleic acid which codes for a protein that may befound useful for a mitochondrion. Contrary to prior art techniques, thetechnology of the invention is indeed not limited by the level ofhydrophobicity of the encoded protein.

Said at least one CDS may thus be any nucleic acid coding for amitochondrial protein. This nucleic acid may be a mitochondrial nucleicacid, or a nuclear nucleic acid coding for a mitochondrial protein.

Most preferably said at least one CDS sequence codes for anaturally-occurring functional mitochondrial protein, such as Cox1,Cox2, Cox3, Atp6, Atp8, Cytb, Nd1, Nd2, Nd3, Nd4, Nd41, Nd5, Nd6.

Preferably, said at least one CDS sequence is the sequence of anaturally-occurring mitochondrial nucleic acid, recoded in accordancewith the universal genetic code.

The mitochondrial nucleic acids use a mitochondrial genetic code whichis slightly different from the universal genetic code that is used bynuclear nucleic acids.

When the protein to be imported into said mitochondrion corresponds to anaturally-occurring mitochondrial protein, the naturally-occurring formof its nucleic acid sequence follows the mitochondrial genetic code.

When such a mitochondrial nucleic acid has to be inserted in the vectorof the invention, the mitochondrial nucleic acid sequence has to berecoded in accordance with the universal genetic code, as the vectordirects a co-translational importation process from nucleus tomitochondrion. Hence, a nuclear-encoded version of the mitochondrialnucleic acid sequence has to be created. This nuclear-encoded versioncan be produced by codon substitution in the mitochondrial nucleic acid,so as to replace those codons which are read by the mitochondrialgenetic system with codons of the universal genetic code. For example,the mammalian UGA codon directs insertion of a tryptophan inmitochondria, but is a stop codon in the nuclear genetic code.Therefore, the UGA codon of a mitochondrial nucleic acid has to bereplaced with UGG which codes for tryptophan in the universal geneticcode.

TABLE 4 universal vs. mitochondrial genetic code codon Universal codeHuman mitochondrial code UGA Stop Trp AGA Arg Stop AGG Arg Stop AUA IleMet

Codon usage in mitochondria vs. the universal genetic code is describedin Lewin, Genes V, Oxford University Press; New York 1994, the contentof which being incorporated by reference.

Codon substitutions notably include:

-   -   UGA to UGG,    -   AGA to UAA, UAG or UGA,    -   AGG to UAA, UAG or UGA,    -   AUA to AUG, CUG or GUG,    -   AUU to AUG, CUG or GUG.

Said at least one CDS sequence may e.g. a nucleic acid sequence codingfor Atp6, or Nd1, or Nd4, such as a nucleic acid sequence of ATP6, or ofND1, or of ND4, recoded in accordance with the universal genetic code(e.g. a sequence of SEQ ID NO:27, NO:28 or NO:29, see FIG. 9).

Said at least one CDS sequence may e.g. a nucleic acid sequence anucleic acid sequence coding for Cox1, Cox2, Cox3, Atp8, Cytb, Nd2, Nd3,Nd41, Nd5, Nd6, such as a nucleic acid sequence of COX1, COX2, COX3,ATP8, Cytb, ND2, ND3, ND41, ND5, ND6.

The description of the thirteen naturally-occurring mitochondrialnucleic acids can be found in Andrew et al. 1999 (Nat Genet. 1999October; 23(2): 147).

Preferably, said recoding is made taking into account the preferredusage codon of said mammalian cell, and most preferably taking intoaccount the human preferred usage codon.

When recoding mitochondrial nucleic acid according to the universalgenetic code, it is according to the present invention very advantageousto take into account the preferred codon usage of the subject orpatient, to which the vector or nucleic acid of the invention is to beadministered.

Preferred codon usage principles, as well as examples of preferred codonusage for various organisms can e.g. be found in Klump and Maeder, 1991(Pure & Appl. Chem., vol. 63, No. 10, pp. 1357-1366 “the thermodynamicbasis of the genetic code”), the content of which is herein incorporatedby reference. An illustrative preferred codon usage for human beings isshown in Table 3 below (see example 2).

Said at least one CDS sequence may e.g. the nucleic acid sequence of SEQID NO:28 or of SEQ ID NO:29 (i.e. a nucleic acid sequence of ND1 or ofND4, recoded in accordance with the universal genetic code, and takinginto account the human preferred usage codon).

The vector of the invention may e.g. comprise:

-   -   at least one SOD2 MTS nucleic acid sequence and at least one        SOD2 3′UTR, or    -   at least one COX10 MTS nucleic acid sequence and at least one        COX10 3′UTR, or    -   any combination of these MTS nucleic acid sequences and 3′UTR        that the skilled person may find appropriate.

Such a vector may e.g. comprise a recoded ATP6, ND1 or ND4 as CDS.

The vector of the invention may e.g. comprise at least one sequence ofSEQ ID NO:21 (COX10 MTS-re-coded ATP6-COX10 3′UTR), SEQ ID NO:22 (SOD2MTS-re-coded ATP6-SOD2 3′UTR), SEQ ID NO:25 (COX10 MTS-re-codedND1-COX10 3′UTR), SEQ ID NO:25 (COX10 MTS-re-coded ND4 COX10 3′UTR).

Alternatively, said at least one CDS sequence may be the nucleic acidsequence of a nuclear nucleic acid which encodes a functionalmitochondrial protein, e.g., a naturally-occurring nuclear nucleic acidwhich encodes a functional mitochondrial protein.

More particularly, said at least one nuclear nucleic acid can be anuclearly-transcribed mitochondrially-targeted mRNA, or the cDNAsequence of such a mRNA, or the DNA sequence coding for such a mRNA.

More particularly, said at least one nuclear nucleic acid can be anuclearly-transcribed mRNA which is not mitochondrially-targeted, or thecDNA sequence of such a mRNA, or the DNA sequence coding for such amRNA.

Said vector may further comprise one or several expression controlsequences. The selection of suitable expression control sequences, suchas promoters is well known in the art, as is the selection ofappropriate expression vectors (see e.g. Sambrook et al. “MolecularCloning: A laboratory Manual”, 2^(nd) ed., vols. 1-3, Cold Spring HarborLaboratory, 1989, the content of which is herein incorporated byreference).

Said vector may thus further comprise at least one promoter operablylinked to said at least one MTS sequence, said at least one CDSsequence, said at least one 3′ sequence.

Said promoter may e.g. be a constitutive promoter, such as e.g. a CMVpromoter.

Said vector may further comprise a termination site.

Said vector may further comprise one of several of the followingexpression control sequences: insulators, silencers, IRES, enhancers,initiation sites, termination signals.

Said vector may further comprise an origin of replication.

Preferably, said promoter and said origin of replication are adapted tothe transduction or infection of animal or human cells, preferably tothe transduction or infection of human cells.

Said vector can e.g. a plasmid, or a virus, such as an integrating viralvector, e.g. a retrovirus, an adeno-associated virus (AAV), or alentivirus, or is a non-integrating viral vector, such as an adenovirus,an alphavirus, a Herpes Simplex Virus (HSV).

Said vector may further comprise a nucleic acid coding for a detectablemarker, such as a FLAG epitope or green fluorescent protein (GFP)

The present invention also relates to a process for the production of avector of the invention, which comprises:

-   -   providing a vector, and depleting from its original 3′UTR, if        any,    -   inserting in this vector at least one MTS nucleic acid sequence,        at least one CDS sequence, and at least one 3′ sequence as        above-described.

As already-mentioned, said vector should preferably not comprise anysequence corresponding to the 3′UTR of a nuclearly-encoded butnon-mitochondrially-targeted mRNA. To the best of the inventors'knowledge, all commercially-available vectors contain such aninappropriate 3′UTR; according to the present invention, such a 3′UTRshould hence be removed from the vector. It may e.g. be replaced by anappropriate 3′ sequence corresponding to a nuclearly-encoded andmitochondrially-targeted mRNA.

Nucleic Acid Construct of the Invention:

The nucleic acid construct which is carried by the vector of theinvention is also encompassed by the present invention. The presentinvention more particularly relates to a non-naturally occurring nucleicacid construct.

A non-naturally occurring nucleic acid construct of the inventioncomprises:

-   -   at least one mitochondrion-targeting nucleic acid sequence        (referred to as MTS nucleic acid sequence),    -   at least one nucleic acid sequence which encodes said protein in        accordance with the universal genetic code (referred to as CDS        sequence), and    -   at least one 3′ nucleic acid sequence, which is located in 3′ of        said at least one MTS nucleic acid sequence and of said at least        one CDS sequence.

Said at least one MTS nucleic acid sequence is:

-   -   the MTS RNA sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, such as the MTS RNA sequence of a        naturally-occurring nuclearly-encoded mitochondrially-targeted        mRNA, or    -   the cDNA sequence of such a RNA, or    -   a DNA sequence coding for such a MTS RNA sequence, or    -   a conservative variant or fragment of such a RNA or DNA MTS        sequence, which derives therefrom by deletion and/or        substitution and/or addition of one or several nucleotides, but        has retained a mitochondrion-targeting function.

Said at least one 3′ nucleic acid sequence is:

-   -   the 3′UTR sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, such as the 3′UTR sequence of a        naturally-occurring nuclearly-encoded mitochondrially-targeted        mRNA, or    -   the cDNA sequence of such a RNA, or    -   a DNA sequence coding for such a 3′UTR sequence, or    -   a conservative variant or fragment of such a RNA or DNA 3′UTR        sequence, which derives therefrom by deletion and/or        substitution and/or addition of one or several nucleotides, and        which, when replacing the wild-type 3′UTR of said        nuclearly-encoded mitochondrially-targeted mRNA, still allows        for a mitochondrial targeting of the resulting mRNA.

It may be provided that, when said at least one MTS nucleic acidsequence is the MTS RNA sequence of a naturally-occurringnuclearly-encoded mitochondrially-targeted mRNA, or the cDNA sequence ofsuch a mRNA, or a DNA sequence coding for such a MTS RNA sequence inaccordance with the universal genetic code, said at least one nucleicacid CDS sequence is not the CDS of this naturally-occurringnuclearly-encoded mitochondrially-targeted mRNA.

It may be provided that, when said at least one 3′ nucleic acid sequenceis the 3′UTR sequence of a naturally-occurring nuclearly-encodedmitochondrially-targeted mRNA, or the cDNA sequence of such a mRNA, or aDNA sequence coding for such a 3′UTR sequence, said at least one CDSsequence is not the CDS of this naturally-occurring nuclearly-encodedmitochondrially-targeted m RNA.

It may be provided that, when said at least one MTS nucleic acidsequence and said 3′ nucleic acid sequence, respectively, are the MTSand 3′UTR sequences of a naturally-occurring nuclearly-encodedmitochondrially-targeted mRNA, or the cDNA sequences of such a mRNA, ora DNA sequence coding for such a mRNA sequence, then said at least oneCDS sequence is not the CDS of this naturally-occurringnuclearly-encoded mitochondrially-targeted mRNA.

Preferably, said nucleic acid construct does not comprise any sequencewhich would be identical to:

-   -   the 3′UTR of a naturally-occurring mRNA which is a        nuclearly-transcribed but not-mitochondrially-targeted mRNA, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding for such a naturally-occurring mRNA 3′UTR        in accordance with the universal genetic code.

The resulting nucleic acid construct does not use a post-translationimportation pathway, but uses a co-translation importation pathway fromnucleus to said mitochondrion.

The present invention more particularly relates to a non-naturallyoccurring nucleic acid construct which comprises:

-   -   at least one mitochondrion-targeting nucleic acid sequence        (referred to as MTS nucleic acid sequence),    -   at least one nucleic acid sequence which encodes said protein in        accordance with the universal genetic code (referred to as CDS        sequence), and    -   at least one 3′ nucleic acid sequence, which is located in 3′ of        said at least one MTS nucleic acid sequence and of said at least        one CDS sequence,        wherein said at least one MTS nucleic acid sequence is:    -   the cDNA sequence of the MTS RNA sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, or    -   a conservative variant or fragment of such a cDNA sequence,        which derives therefrom by deletion and/or substitution and/or        addition of one or several nucleotides, but has retained a        mitochondrion-targeting function,        wherein said at least one 3′ nucleic acid sequence is:    -   the cDNA sequence of the 3′UTR sequence of a nuclearly-encoded        mitochondrially-targeted mRNA, or    -   a conservative variant or fragment of such a cDNA 3′UTR        sequence, which derives therefrom by deletion and/or        substitution and/or addition of one or several nucleotides, and        which, when replacing the wild-type 3′UTR of said        nuclearly-encoded mitochondrially-targeted mRNA, still allows        for a mitochondrial targeting of the resulting mRNA,        provided that, when said at least one MTS nucleic acid sequence        and said at least one 3′ nucleic acid sequence, respectively,        are the MTS and 3′UTR sequences of a naturally-occurring        nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA        sequences of such a mRNA, or a DNA sequence coding for such a        mRNA sequence, then said at least one CDS sequence is not the        CDS of this naturally-occurring nuclearly-encoded        mitochondrially-targeted mRNA, and        wherein said nucleic acid construct does not comprise any        sequence which would be identical to:    -   the 3′UTR of a naturally-occurring mRNA which is a        nuclearly-transcribed but not-mitochondrially-targeted mRNA, or    -   the cDNA sequence of such a 3′UTR sequence, or    -   a DNA sequence coding for such a naturally-occurring mRNA 3′UTR        in accordance with the universal genetic code.

Each and every feature, herein and above described for the MTS, CDS, 3′nucleic acid sequences in relation with the vector of the invention, andnotably those features relating to the MTS, CDS, 3′ nucleic acidsequences, of course applies mutatis mutandis to the nucleic acidcontruct of the invention, and more particularly to the non-naturallyoccurring nucleic acid contruct of the invention.

Hence, it notably follows that:

-   -   a MTS nucleic acid sequence of said nucleic acid construct can        be the MTS nucleic acid sequence of ACO2, or of SOD2, or of        ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2,        or of COX10;    -   a 3′ nucleic acid sequence of said nucleic acid construct can        be:        -   the 3′UTR sequence of ACO2, or of SOD2, or of ATP5b, or of            UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of            COX10, or of AK2, or        -   the cDNA sequence of such a 3′UTR sequence, or        -   a DNA sequence coding for such a 3′UTR sequence in            accordance with the universal genetic code; and that    -   illustrative nucleic acid constructs of the invention comprise        or consist of a sequence of SEQ ID NO:21 (COX10 MTS-re-coded        ATP6-COX10 3′UTR), and/or of SEQ ID NO:22 (SOD2 MTS-re-coded        ATP6-SOD2 3′UTR), and/or of SEQ ID NO:25 (COX10 MIS-re-coded        ND1-COX10 3′UTR), and/or of SEQ ID NO:26 (COX10 MTS-re-coded        ND4-COX10 3′UTR).

Said non-naturally occurring nucleic acid construct may be transfectedin a cell in the form of naked DNA, or in the form of a plasmid. Anytransfection technology which is found convenient by the skilled personis convenient. The skilled person may e.g. proceed by electroporation,DEAE Dextran transfection, calcium phosphate transfection, cationicliposome fusion, creation of an in vivo electrical field, DNA-coatedmicroprojectile bombardment, ex vivo gene therapy, and the like.

Said non-naturally occurring nucleic acid construct may of coursealternatively and/or complementarily be inserted into a vector, such asa viral vector.

The vector and nucleic acid construct of the invention are useful fornucleic acid therapy, e.g. to reverse a cellular dysfunction caused by amutation in nucleic acid coding for a mitochondrial protein. They enableto restore a protein function in a cell.

Engineered Cell:

The present invention also relates to an engineered cell which has beentransduced or infected by a vector according to the invention, and/ortransfected by a nucleic acid construct according to the invention.

Preferably, said engineered cell is an engineered animal or human cell,most preferably to a mammalian engineered cell, still more preferably toan engineered human cell.

Said engineered cell may e.g. be a bone-marrow cell, a clonal cell, agerm-line cell, a post-mitotic cell, such as a cell of the centralnervous system; a neuronal cell, a retinal ganglion cell, a progenitorcell; or a stem cell, a hematopoietic stem cell, a mesenchymal stemcell. Preferably, said engineered cell is a neuronal cell, a retinalganglion cell.

Said transduction, infection or transfection may be impleted by anymeans available to the skilled person, e.g. by electroporation, DEAEDextran transfection, calcium phosphate transfection, cationic liposomefusion, creation of an in vivo electrical field, DNA-coatedmicroprojectile bombardment, injection with a recombinantreplicative-defective virus, homologous recombination, ex vivo genetherapy, a viral vector, naked DNA transfer, and the like.

Said engineered cell may e.g. be a cell, such as a neuronal cell,collected from a patient suffering from a disease related to amitochondrial dysfunction. The vector and nucleic acid construct of theinvention can indeed be used for ex vivo cell therapy.

Pharmaceutical Compositions and Applications:

The present invention also relates to a pharmaceutical compositioncomprising at least one vector according to the invention, or at leastone nucleic acid construct according to the invention, or at least oneengineered mammalian cell according to the invention.

The present invention also relates to a drug comprising at least onevector according to the invention, or at least one nucleic acidconstruct according to the invention, or at least one engineeredmammalian cell according to the invention.

The compositions of the present invention may further comprise at leastone pharmaceutically and/or physiologically acceptable vehicle (diluent,excipient, additive, pH adjuster, emulsifier or dispersing agent,preservative, surfactant, gelling agent, as well as buffering and otherstabilizing and solubilizing agent, etc.).

Appropriate pharmaceutically acceptable vehicles and formulationsinclude all known pharmaceutically acceptable vehicles and formulations,such as those described in “Remington: The Science and Practice ofPharmacy”, 20^(th) edition, Mack Publishing Co.; and “PharmaceuticalDosage Forms and Drug Delivery Systems”, Ansel, Popovich and Allen Jr.,Lippincott Williams and Wilkins.

In general, the nature of the vehicle will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise, in addition to the one or more contrast agents,injectable fluids that include pharmaceutically and physiologicallyacceptable fluids, including water, physiological saline, balanced saltsolutions, buffers, aqueous dextrose, glycerol, ethanol, sesame oil,combinations thereof, or the like as a vehicle. The medium also maycontain conventional pharmaceutical adjunct materials such as, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. The carrier andcomposition can be sterile, and the formulation suits the mode ofadministration.

The composition can be e.g., be in the form of a liquid solution,suspension, emulsion, capsule, sustained release formulation, or powder.

The pharmaceutical composition and drug of the invention are useful forthe therapeutic and/or palliative and/or preventive treatment of adisease, condition, or disorder related to a defect in activity orfunction of mitochondria.

A mitomap is available on the MITOMAP website; this site notablyprovides with a list of mitochondrial disease-associated mutations.

Scientific publication review relating to mitochondrial disease,condition, or disorder notably comprise Carelli et al. 2004 (Progress inRetinal and Eye Research 23: 53-89), DiMauro 2004 (Biochimica etBiophysica Acta 1659:107-114), Zeviani and Carelli 2003 (Curr OpinNeurol 16:585-594), and Schaefer et al. 2004 (Biochimica et BiophysicaActa 1659: 115-120), the contents of which being herein incorporated byreference.

Diseases, conditions or disorders related to a defect in mitochondriaactivity or function notably comprise myopathies and neuropathies, suchas optic neuropathies. Examples of mitochondrial diseases, conditions ordisorders comprise: aging, aminoglycoside-induced deafness,cardiomyopathy, CPEO (chronic progressive external ophtalmoplegia),encephalomyopathy, FBSN (familial bilateral stritial necrosis), KS(Kearns-Sayre) syndrome, LHON (Leber's hereditary optic neuropathy),MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke-like episodes), MERRF (myoclonic epilepsy with stroke-likeepisodes), MILS (maternally-inherited Leigh syndrome), mitochondrialmyopathy, NARP (neropathy, ataxia, and retinis pigmentosa), PEO, SNE(subacute necrotizing encephalopathy).

Optic neuropathies notably comprise:

-   -   Leber's hereditary optic neuropathy (LHON), which involves one        or several point mutation(s) in mitochondrial DNA, more        particularly point mutation(s) in the ND1 and/or ND4 and/or ND6        gene(s), such as G3460A (ND1 mutation), G11778A (ND4 mutation),        T144840 (ND6 mutation),    -   the dominant optic atrophy (DOA), also known as Kjer's optic        neuropathy, which involves a defect in nuclear gene OPA1,    -   the FBSN, MILS and NARP, which are the result of a mutation in        the MTATP6 gene (defective ATP synthesis), which can be        corrected by restoring the activity of function of ATP6.

Leber's hereditary optic neuropathy (LHON) was the first maternallyinherited disease to be associated with point mutations in mitochondrialDNA and is now considered the most prevalent mitochondrial disorder. Thepathology is characterized by selective loss of retinal ganglion cellsleading to central vision loss and optic atrophy, prevalently in youngmales. It is a devastating disorder with the majority of patientsshowing no functional improvement and remaining within the legalrequirement for blind registration. Other clinical abnormalities havealso been reported in LHON patients. These include postural tremor,peripheral neuropathy, non-specific myopathy, movement disorders andcardiac arrhythmias [8]. The three most common pathogenic mutations fromLHON affect complex I ND1 and/or ND4 and/or ND6 genes with the doubleeffect of lowering ATP synthesis and increasing oxidative stresschronically.

Each of said disease, condition or disorder could be corrected byrestoring activity or function of the mutated DNA.

Example 1 below illustrates the rescue of an ATP6 activity or functionwith a vector and nucleic acid of the invention. Example 2 belowillustrates the rescue of a ND1 and ND4 activity or function with avector and nucleic acid of the invention (fibroblasts collected fromLHON patients).

The pharmaceutical compositions or drugs of the invention are moreparticularly intended for the therapeutic and/or palliative and/orpreventive treatment of a myopathy or of an optic neuropathy, such asLHON, DOA, FBSN, MILS or NARP.

The present invention relates to the use of at least one vector ornucleic acid construct for in vivo or ex vivo therapy of a subject orpatient in need of a therapeutic, palliative or preventive treatment ofa disease, condition, or disorder related to a defect in activity orfunction of mitochondria.

The present invention also relates to the use of at least one vector ornucleic acid construct of the invention, or of at least one engineeredcell of for the treatment of a disease, condition, or disorder relatedto a defect in activity or function of mitochondria, and moreparticularly for the production of a composition, pharmaceuticalcomposition or drug intended for the treatment of such a diseasecondition, or disorder.

The present invention more particularly relates to a method for thetherapeutic and/or palliative and/or preventive treatment of a disease,condition, or disorder related to a defect in activity or function ofmitochondria, which comprises:

-   -   administering to a subject or patient in need thereof a vector        and/or a nucleic acid construct and/or an engineered cell of the        invention, in a quantity effective for the therapeutic and/or        palliative and/or preventive treatment of said subject or        patient,    -   ex vivo treating cells collected from a subject or patient in        need thereof, and returning the treated cells to the subject or        patient.

The term “comprising”, which is synonymous with “including” or“containing”, is open-ended, and does not exclude additional, unrecitedelement(s), ingredient(s) or method step(s), whereas the term“consisting of” is a closed term, which excludes any additional element,step, or ingredient which is not explicitly recited.

The term “essentially consisting of” is a partially open term, whichdoes not exclude additional, unrecited element(s), step(s), oringredient(s), as long as these additional element(s), step(s) oringredient(s) do not materially affect the basic and novel properties ofthe invention.

The term “comprising” (or “comprise(s)”) hence includes the term“consisting of” (“consist(s) of”), as well as the term “essentiallyconsisting of” (“essentially consist(s) of”). Accordingly, the term“comprising” (or “comprise(s)”) is, in the present application, meant asmore particularly encompassing the term “consisting of” (“consist(s)of”), and the term “essentially consisting of” (“essentially consist(s)of”).

Each of the relevant disclosures of all references cited herein isspecifically incorporated by reference.

The present invention is illustrated by the following examples, whichare given for illustrative purposes only.

EXAMPLES Example 1: Allotopic Expression of the ATP6 Mitochondrial Geneis Significantly Improved by the Localization of its mRNAs to theSurface of Mitochondria Leading to an Efficient Import of the PrecursorAbstract

It is clear that impairment of mitochondrial energy metabolism is thekey pathogenic factor in a growing number of neurodegenerativedisorders. With the discovery of mtDNA mutations, the replacement ofdefective genes became an important goal for mitochondrial geneticistsworldwide. Unfortunately, before the present invention, it was still notpossible to introduce foreign genes into the mitochondria of mammaliancells.

To circumvent this problem, allotopic expression in the nucleus of genesencoded by mitochondrial DNA (mtDNA), became an attractive idea.However, for most is mitochondrial genes tested, there were importantlimitations related to the high hydrophobicity of the correspondingproteins, which impedes their mitochondrial translocation.

We herein elucidate the mechanisms that enable the delivery of mRNAsencoding mitochondrial proteins to the organelle surface, anddemonstrate that this delivery depends on two sequences: the regioncoding for the mitochondrial targeting sequence (MTS) and the 3′UTR.mRNA sorting to mitochondrial surface permits to optimize allotopicapproach, by enhancing the mitochondrial import efficiency of theprecursor synthesized in the cytosol. As an illustration of thismechanism, we have chosen to utilize the sequence coding for the MTS andthe 3′UTR of two nuclear genes encoding mitochondrial proteins: COX10and SOD2 associated to a recoded mitochondrial ATP6 gene. Indeed, COX10and SOD2 mRNAs localize to the mitochondrial surface in HeLa cells. HeLacells transfected with these constructions express an Atp6 protein whichis successfully delivered to the mitochondria. Hence, we have been ableto optimize the allotopic approach for Atp6, and our procedure will nextbe tried to rescue mitochondrial dysfunction in patients presenting ATP6mutations.

Introduction

To examine the possibility that allotopic expression of mtDNA genescould be optimized by a targeted localization of the mRNA to themitochondrial surface, we have chosen to utilize the sequences codingfor the MTS and the 3′UTR of two nuclear genes encoding mitochondrialproteins: COX10 and SOD2 associated to an reengineered nucleus-localizedATP6 gene. COX10 encodes a highly hydrophobic protein of the innermitochondrial membrane, its mRNAs localizes to the mitochondrial surface[5]. SOD2 encodes a mitochondrial protein involved in detoxification,its mRNA, as COX10 mRNA, localizes to the mitochondrial surface [5] anda recent report described that in HeLa cells, its 3′UTR is associated tothe mitochondrial surface via the Akap121 protein [6]. The ability tosynthesize and direct the Atp6 protein to mitochondria was examined inHela cells for 4 plasmids: two of them only contain the mts of COX10 orSOD2, and the two other possess both the MTS and the 3′UTR of each gene.Hybrid mRNAs were detected for each construction in both transiently andstably transfected cells. Further, Atp6 protein was also visualized byindirect immunofluorescence associated to the surface of mitochondria.Mitochondria isolated from transfected cells were examined for thepresence of Atp6 protein. Remarkably, hybrids mRNAs possessing both theMTS and the 3′UTR of either COX10 and SOD2 allow the synthesis of apolypeptide which is imported in a highly efficient way from the cytosolinto the mitochondria. Thus, the strategy of directing a hybrid mRNA tothe mitochondrial surface significantly improves the feasibility of theallotopic approach for mitochondrial genes.

Material and Methods:

Plasmid Construction:

The full-length ATP6 mitochondrial gene was reengineered after theproduction of the 677 pb product by RT-PCR (Superscript III one stepRT-PCR Platinium Taq HiFi, Invitrogen), using total RNA from HeLa cells.The KR product obtained was cloned in the PCR 2.1-Topo vector(Invitrogen, Life technologies). In this vector, we recoded 11non-universal codons in the ATP6 gene by four rounds of in vitromutagenesis (Quik change Multi site-directed mutagenesis kit;Stratagene, La Jolla, Calif.). Six oligonucleotide primers were designedto alter AUA codons to AUG and UGA to UGG (Table 1).

TABLE 1 In vitro mutagenesis of the ATP6 mitochondrial gene Length Namesequence (bp) ATP6.1 CAATGGCTAATCAAACTAACCTCAAAACAAATGA 78TGACCATGCACAACACTAAAGGACGAACCTGGTC TCTTATGCTA (SEQ ID NO: 1) ATP6.2TCTATGAACCTAGCCATGGCCATCCCCTTATGGG 54CGGGCACAGTGATTATGGGC (SEQ ID NO: 2) ATP6.3CCCATGCTAGTTATTATCGAAACCATCAGCCTAC 51 TCATTCAACCAATGGCC (SEQ ID NO: 3)ATP6.4 ACCCTAGCAATGTCAACCATTAAC (SEQ ID 24 NO: 4) ATP6.5ACTAAAGGACGAACCTGGTCTCTTATGCTAGTAT 42 CCTTAATC (SEQ ID NO: 5) ATP6.6ACACCAACCACCCAACTATCTATGAACCTAGCCA 42 TGGCCATC (SEQ ID NO: 6)

The intermediate construct was sequenced for accuracy. To this recodedATP6, we appended in frame either the MTS of COX10 or 5002, obtained byRT-PCR using total RNA from HeLa cells (Superscript III one step RT-PCRPlatinium Taq HiFi; Invitrogen, Life technologies). For COX10 weamplified the sequence corresponding to the first 28 amino acids, forSOD2 the sequence coding for the first 30 amino acids. Oligonucleotideprimers used for the amplification include at its 3′ extremity a Sal1restriction site for the subsequent cloning in frame with thereengineered ATP6 gene which possesses a Sal1 restriction site at its 5′extremity (Table 2).

TABLE 2 Oligonucleotides primers for RT-PCR analysis RT-PCR productlength Name 5′ Primer (5′-3′) 3′Primer (5′-3′) (bp) ATP6 GTCGACCGCATGACCGGGCGGCCGCTGT 677 ORF ACGAAAATCTGTTC GTTGTCGTGCAGGTA GCTTCATTCATTGAGGCTTAC (SEQ ID NO: 7) (SEQ ID NO: 8) MTS CGCTCTAGAATGGGCGGTCGACTTCAAG 84 COX10 CCGCATCTCCGCA ATACCAGACAGAGCC CACTCTC TCC(SEQ ID NO: 9) (SEQ ID NO: 10) ′UTR CCCGATCGGAGCA CGCACGCGTAAAGCT 1429COX10 CTGGGACGCCCAC TCTACAAATGTGAAGG CGCCCCTTTCCC CTGTAACA(SEQ ID NO: 11) (SEQ ID NO: 12) MTS CGCTCTAGAATGTT GTCGACCGCGTCGGG 90SOD2 GAGCCGGGCAGTG GAGGCTGTGCTTCTG TGCGGC CCT (SEQ ID NO: 13)(SEQ ID NO: 14) 3′UTR ACCACGATCGTTAT CGCACGCGTCAATCA 215 SOD2GCTGAGTATGTTAA CACAAAGCATTTACTA GCTCTTTA TTTTC (SEQ ID NO: 15)(SEQ ID NO: 16) COX6c ATGGCTCCCGAAG CTGAAAGATACCAGC 250 TTTTGCCAAAACCTCTTCCTCATCTC (SEQ ID NO: 17) (SEQ ID NO: 18) SOD2 CGACTACGGCGCCCTGGAACCTCACA TCAACGC (SEQ ID NO: 58)

The final sequences of the fusion ATP6 genes were checked for accuracy,and inserted in the pCMV-Tag 4A vector (Stratagene, La Jolla Calif.),which will direct the synthesis of the protein via the CMV promoter andits detection by the presence of a FLAG epitope tag appended to theC-terminal region of Atp6. To obtain hybrid mRNAs which will alsocontain the 3′UTR of COX10 or SOD2 genes we replaced the SV40 polyAsignal present in the pCMV-Tag 4A vector (positions 1373-1679) usingPvu1 and Mlu1 restriction enzymes, by the 1429 bp of the full-lengthCOX10 3′UTR or the 215 pb of the SOD2 3′UTR. Both 3′UTR were firstobtained by RT-PCR using RNAs purified from HeLa cells and specificoligonucleotide primers containing Pvu1 and Mlu1 restriction sites ateach end (cf. Table 2 above). PCR fragments were first cloned in the PCR2.1-Topp vector (Invitrogen, Life technologies) and sequenced to verifythat no mistakes were generated before subcloning in the pCMV-Tag 4Avector. The four final constructs were entirely sequenced for accuracyusing specific oligonucleotide primers to verify the full-lengthsequences of either the fusion ATP6 genes or the 3′UTR regions appendedto them. Final sequences inserted in the pCMV-Tag 4A vectors are shownin FIG. 1B.

The sequence of recoded ATP6 (SEQ ID NO: 27) is shown on FIG. 9. The MTSand 3′UTR sequences of COX10 and SOD2 are shown on FIG. 10 (SEQ ID NO:30, 47, 31 and 60).

Cell Culture and Transfection:

We cultured HeLa cells with RPMI medium complemented with 10% of foetalbovine serum (Gibco, Invitrogen), gentamicin (0.01%), 2 mM glutamine,optionally with pyruvate (e.g., 2.5 mM), optionally with antibiotics(such as 100 u/mL penicillin, 100 μg/mL streptomycin). They weretransfected with FuGENE 6 transfection reagent as recommended by themanufacturer (Roche Biochemicals, Indianapolis). Briefly, monolayer Helacells were seeded a day before transfection at 50% confluence, so thenext day they will be at approximately 80% confluence, the cells wereplated in a medium without antibiotics. 2 microgrammes of differentplasmids purified with Quiagen plasmid midi kit (Quiagen; Valencia,Calif.) were used. Between 48 to 60 hr later, 80% of the transfectedcells were used either for immunochemistry analyses or RNA andmitochondria extractions. The remaining 20% of cells were selected forneomycine, G418, resistance (selectable marker present in the pCMV-Tag4A vector) at a final concentration of 1 mg/ml. Stable clones wereexpanded for several weeks, immunochemistry analyses were performed.Mitochondria were also isolated to determine the import ability of theAtp6 protein.

Immunocytochemistry:

Coverslips were placed on the bottom of 24-well dishes and HeLa cellsseeded at approximately 50% confluence (80000 cells). 60 hours aftertransfection, cells were fixed with 2% paraformaldehyde in PBS for 15min and processed for indirect immunofluorescence. Afterpermeabilization of the cells for 5 min with Triton 1% in PBS, cellswere incubated for one hour in PBS with 1% BSA before the addition ofthe primary antibodies: mouse monoclonal anti-Flag M2 antibodies(Stratagene, La Jolla Calif.) or mouse monoclonal anti-ATP synthasesubunit beta (Molecular Probes, Invitrogen). Both antibodies were usedat a final concentration of 1 microgramme/ml. The incubation withprimary antibodies was performed for either 2 hr at room temperature orovernight at 4° C. After washing the primary antibody three times fivemin with PBS, cells were incubated with the secondary antibody: labeledgoat-anti-mouse□IgG Alexa Fluor 488 (Molecular Probes, Invitrogen). Thisantibody was used in 1% BSA-PBS at 1:600 dilution and placed on the topof the coverslips for two hr. The cells were, subsequently, washed oncein PBS for 5 min. For DNA and mitochondria staining, a second wash wasperformed with 0.3 microgrammes/ml of DAPI (Sigma, Saint Louis, Mich.)and 100 nM of MitoTracker Deep Red 633 (Molecular probes, Invitrogen)for 20 min. A last 10 min wash was performed in PBS and the coverslipswere mounted using Biomeda Gel/Mount. Immunofluorescence was visualizedin a Leica DM 5000 B Digital Microscope. Digital images were acquiredand processed with the MetaVue imaging system software.

Mitochondria Isolation and Western Blot Analysis:

Between 20 to 40 millions or 100 millions of transiently or stablytransfected HeLa cells were treated with trypsin (Gifco, Invitrogen) for5 min and spin down. One wash in PBS was performed. The pellets wereresuspended in 10 ml of homogenization buffer: 0.6 Mannitol, 30 mMTris-HCl PH 7.6, 5 mM MgAc and 100 mM KCl, 0.1% fatty acid-free bovineserum albumin (BSA), 5 mM beta-mercaptoethanol and 1 mM PMFS. To theresuspended cells 0.01% of digitonin was added. After a 4 min incubationon ice the homogenization was performed with 15 strokes in a Dounceglass homogenenizer with a manually driven glass pestle type B.Homogenates are centrifuged for 8 min at 1000 g at 4° C. to pelletunbroken cells and nuclei. Since many mitochondria remain trapped inthis pellet, it was resuspended and rehomogenized again with 5 ml ofhomogenization buffer and 25 additional strokes. Then a second round ofcentrifugation under the same conditions was performed. Bothsupernatants were assembled and centrifuged again to discard any nuclearor cell contaminant. The supernatant obtained was centrifuged at 12000 gat 4° C. for 30 min to pellet mitochondria. Four washes inhomogenization buffer were performed to free the mitochondrial fractionof particles containing membranes, reticulum endoplasmic and proteases.The last two washes were performed in a homogenization buffer devoid ofBSA and PMFS to allow a better estimation of the protein concentrationin the final mitochondrial fraction and its subsequent analysis byproteinase K digestion. Protein concentrations in the extracts weremeasured using the dye-binding assay Bradford. To determine whether Atp6was translocated into the organelle, 15 microgrammes of mitochondrialproteins were treated with 200 microgrammes/ml of proteinase K (PK) at0° C. for 30 min. Samples were then resolved in 4-12 gradient or 12%polyacrylamide SDS-PAGE, and transferred to nitrocellulose. Filters wereprobed with the following antibodies: mouse monoclonal anti-Flag M2antibodies (Stratagene, La Jolla Calif.) which recognizes the nuclearrecoded Atp6 protein in which a flag epitope was appended at itsC-terminus or mouse monoclonal anti-ATP synthase subunit alpha(Molecular Probes, Invitrogen), which recognizes the 65 kDa nuclearencoded alpha-subunit of the ATP synthase, Complex V. Immunoreactivebands were visualized with anti-mouse coupled to horseradish peroxidase(1:10000) followed by ECL Plus detection (Amersham International)according to the manufacturer's instructions.

Five independent mitochondria purifications from cells stablytransfected with either SOD2^(MTS) ATP6-3′UTR^(SV40) or SOD2^(MTS)ATP6-3′UTR^(SOD2) vectors were performed. The amount of precursor andmature forms of ATP6 in mitochondria, as well as the quantities of boththe mature form of ATP6 and ATP □ resistant to PK proteolysis werecompared by densitometric analyses (Quantity One, Biorad softwaresystem). The significance of the differences observed was validated witha paired Student's t-test.

RNA Extraction and RT-PCR Analyses:

Mitochondria extractions were performed as described in the precedentsection, with the following modifications: 400 millions of cells weretreated with 250 μg/ml cycloheximide for 20 minutes at 37° C. To HB wasadded 200 Mg/ml cycloheximide, 500 μg/ml heparine and 1/1000 RNaseinhibitor (rRNasin, Promega). The last pellet of crude mitochondriaassociated with polysomes (M-P) was stored at −80° C. until RNAextraction. Free-cytoplasmic polysomes (F-P) were obtained from thepost-mitochondrial supernatant fraction by sedimentation through a stepgradient of 2 M and 0.5 M sucrose. RNAs from these two fractions, aswell as total RNAs from each stably transfected cell line, were obtainedusing RNeasy Protect Mini kit (Qiagen). Generally, 10 millions of cellsare sufficient to obtain approximately 30 microgrammes of total RNA. Thepresence of the hybrid ATP6 mRNA was examined using primers whichrecognize the first 27 nt of either COX10 or SOD2 MTS and a primer whichrecognize the last 27 nt of the ATP6 ORF. For the pCMV-Tag 4A vectorcontaining both the MTS and the 3′UTR of COX10 or SOD2, we used a primerrecognizing the last 27 nt of each 3′UTR. 100 ng of RNA was used forreverse transcription (cf. Table 1 above). The products were thensubjected to 25 cycles of PCR using Superscript III one step RT-PCRPlatinium Taq kit (Invitrogen). As an internal control a 250 nt fragmentwithin the ORF of COX6c gene, encoding a mitochondrial protein, was alsoamplified. Ten percent of the amplified products were run in agarosegels, and the quantities of amplified products reflecting hybrid ATP6mRNA amount in each preparation was estimated using the Photocapsoftware (Vilber Lourmat; Torcy, France).

Tables 1 and 2 above, and table 9 below, show primer sequences, theexpected sizes of the FOR products, the quantity of RNA used forreverse-transcription and the number of FOR cycles performed.

Densitometric analyses (Quantity One, Bio-Rad software) were performedthe amount of both hybrid ATP6 and SOD2 transcripts in eithermitochondrion-bound polysomes or free-cytoplasmic polysomes. Threeindependent RNA preparations from M-P and F-P fractions were subjectedthree times to RT-PCR analyses.

TABLE 9 RT-PCR Polysomal RNAs product Total RNAs (M-P/F-P) lengthPrimers Quantity Cycle Quantity Cycle mRNA (bp) 5′ Primer 3′Primer (ng)numbers (ng) numbers SOD2^(MTS) 780 MTS ATP6 200 28 250 28 ATP6 SOD2 5′ORF3′ ATP6 677 ATP6 ATP6 50 28 150 20 ORF 5′ ORF 3′ SOD2 785 SOD2 5′3′UTR 100 28 20 20 SOD2 3′ COX6c 250 COX6c 5′ COX6c 3′ 200 28 250 20

Results: Construction of Reengineered Mitochondrial ATP6 Gene forAllotopic Expression

To accomplish allotopic expression we synthesized the full-lengthversion of nuclear-encoded ATP6 mitochondrial gene converting codons AUAto AUG and codons UGA to UGG. Indeed, AUA in the mitochondrial geneticsystem leads to the insertion of a methionine, but according to theuniversal code it is an isoleucine. Additionally, UGA into mitochondriacodes for a tryptophan, whereas in the cytosol it represents a stopcodon. We, therefore, recoded all 11 mitochondrial codons present inATP6 ensuring the accurate translation of the transcript by cytoplasmicribosomes. These alterations were performed by four rounds of in vitromutagenesis using six independent oligonucleotide primers (Table 1) andthe Quik change Multi site-directed mutagenesis kit (Stratagene, LaJolla, Calif.).

The concept of allotopic approach has important implications for thedevelopment of therapies to patients with mitochondrial DNA mutations.However, up today a major obstacle remains to be overcome and is thetargeting of the recoded protein to mitochondria. We then decided toforce the localization of the recoded ATP6 mRNA to the mitochondrialsurface. The rationale behind this specific mRNA targeting is to allow aco-translational import mechanism which will maintain the precursor inan import competent conformation impeding its aggregation before orduring translocation through the TOM (Translocase of the outer membrane)and TIM (Translocases of the inner membrane) import complexes. Twosequences within mRNAs are believed to be involved in their localizationto the mitochondrial membrane: the sequence coding for the MTS and the3′UTR. We have chosen two nuclearly-encoded mitochondrial genes, whichmRNAs are preferentially localized to the surface of mitochondria inHeLa cells: COX10 and SOD2 [5]. Interestingly, the SOD2 mRNA has beenshown to be associated to the mitochondrial surface via its 3′UTR andthe Akap121 protein.

We therefore, obtained four different plasmids.

Two contain either the MTS of COX10 or the sequence encoding the first30 amino acids of SOD2 (=the 20 amino acids of the MTS sequence of SOD2,and the ten consecutive amino acids that follows within the SOD2sequence, i.e., fragment 1-30 of SEQ ID NO:49), in frame with the AUGcodon of the recoded ATP6 gene (COX10 MTS-recoded ATP6-SV40 3′ UTR; SOD2MTS-recoded ATP6-SV40 3′ UTR). In these plasmids, the SV40 polyA signalfunctions as the 3′UTR.

The other two combine both the MTS and the 3′UTR of COX10 and SOD2respectively, and do not comprise the cytosolic 3′UTR of SV40 (COX10MTS-recoded ATP6-COX10 3′ UTR; SOD2 MTS-recoded ATP6-SOD2 3′ UTR).

FIGS. 1A and 1B illustrate the constructs obtained and the full-lengthsequences inserted in the pCMV-Tag 4A vector than we named respectively:COX10 MTS-nATP6, SOD2 MTS-nATP6 and COX10 MTS-nATP6-COX10 3′UTR and SOD2MTS-nATP6-SOD2 3′UTR.

Detection of Hybrid ATP6 mRNAs in Transiently and Stably HeLaTransfected Cells

To determine whether transfected cells express hybrid ATP6 mRNAs,steady-state levels of the transcripts were measured in both transientlyand stably transfected cells after the isolation of total RNAs. 100 ngof total RNAs were subjected to RT-KR analyses using specific primeroligonucleotides for hybrid ATP6 mRNA. COX6c gene encoding amitochondrial protein was used as an internal control, with specificprimers allowed the amplification of a 250 bp fragment (FIG. 2). RNAsfrom non-transfected HeLa cells as well as HeLa cells transfected withthe empty pCMV-Tag 4A vector were also tested as negative controls. FIG.2 shows a 780 bp PCR product corresponding to the amplification of thefirst 27 nt of COX10 ORF and the last 27 nt of the ATP6 ORF in cellstransfected with both COX10 MTS-nATP6 and COX10 MTS-nATP6-COX10 3′UTRvectors. Additionally, RNAs isolated from cells transfected with theCOX10 MTS-nATP6-COX10 3′UTR vector amplified a 2374 bp productcorresponding to the entire ATP6 ORF and the full-length COX10 3′UTR.

The results obtained with RNAs purified from transfected cells with SOD2MTS-nATP6 and SOD2 MTS-nATP6-SOD2 3′UTR vectors show that hybrid ATP6transcript was detected as a 780 nt amplified product. Further, the SOD2MTS-nATP6-SOD2 3′UTR region amplified a 1060 bp fragment, correspondingto the entire ATP6 ORF and the full-length SOD2 3′UTR. These resultsindicate that HeLa cells express the reengineered ATP6 gene. Moreover,no significant differences in the steady-state levels of hybrid mRNAswere found by the addition of either COX10 3′UTR or SOD2 3′UTR.

To examine the ability of SOD2 signals associated to the recoded ATP6gene to direct hybrid mRNAs to the mitochondrial surface, we determinedtheir subcellular localization in the four stably cell lines obtained.In this purpose, we isolated RNAs from mitochondrion-bound polysomes(M-P) and free-cytoplasmic polysomes (F-P) and we determined by AT-FORthe steady-state levels of hybrid mRNAs in both polysomal populations(FIG. 12B). As internal controls, the subcellular distribution ofendogenous mitochondrial ATP6. SOD2 and COX6c mRNAs were determined.Endogenous ATP6 mRNA exclusively localized to the mitochondrialcompartment as expected. Besides, endogenous SOD2 mRNA is enriched inmitochondrion-bound polysomes (M-P), whereas COX6c mRNA ispreferentially detected in free-cytoplasmic polysomes (F-P) as we havepreviously observed. The SOD2MTSATP6-3′UTRSOD2 vector directed thesynthesis of a hybrid mRNA that was almost undetectable infree-cytoplasmic polysomes. Hybrid mRNA produced from theSOD2MTSATP6-3′UTRSV40 plasmid was also detected preferentially inmitochondrion-bound polysomes. However, it was also present infree-cytoplasmic polysomes (FIG. 12B). Densitometric analyses wereperformed to determine the amount of both endogenous SOD2 and hybridATP6 mRNAs in each polysomal population examined. SOD2 mRNA signal inmitochondrion-bound polysomes was 85.6%±6.15 in cell lines expressingthe SOD2MTSATP6-3′UTRSV40 plasmid and 82.5%±4.87 in cells expressing theSOD2MTSATP6-3′UTRSOD2 vector. Interestingly, it was found for the ATP6hybrid mRNA that only 72.4%±5.2 localized to the mitochondrial surfacein cells expressing the SOD2MTSATP6-3′UTRSV40 vector. Instead, in cellsexpressing the SOD2MTSATP6-3′UTRSOD2 vector 84.6%±4.7 of the hybrid mRNAlocalized to the mitochondrial surface (FIG. 120). These values weresignificantly different according to the paired Student's t-test(P<0.0034, n=6). Thus, the combination of both the MTS and 3′UTR of SOD2to the reengineered ATP6 gene leads to the synthesis in the nucleus of atranscript which was almost exclusively sorted to the mitochondrialsurface. Indeed, its subcellular distribution is not significantlydifferent to the one of the endogenous SOD2 mRNA.

TABLE 10 ATP6 mRNA signal localized at the ATP6 mRNA within Hela cellsmitochondrial surface the mitochondria With a cytosolic 3′UTR 72.4% ± 50.71 ± 0.12 (SV40 3′ UTR) With a mitochondrial     82.5 ± 4.8 1.28 ±0.24 3′UTR (SOD2 3′UTR), and without any cytosolic 3′UTR

Detection of ATP6 Allotopic Expression in HeLa Cells by IndirectImmunofluorescence

We analyzed the ability of the reengineered ATP6 product to localize tomitochondria in vivo.

For this, we appended a Flag epitope in frame to the C-terminus of theATP6 ORF and we examined stably transfected cells by indirectimmunofluorescence (FIG. 13). HeLa cells transfected with the emptypCMV-Tag 4A vector were used as negative controls and showed a lowdiffused signal in cytoplasm when antibodies to Flag were used (FIG. 13,left panel). Stably transfected cells with either SOD2^(MTS)ATP6-3′UTR^(SV40) or SOD2^(MTS) ATP6-3′UTR^(SOD2) vectors werevisualized by indirect immunofluorescence using antibodies to Flag (FIG.14, left panel) and to ATP synthase subunit α (FIG. 13 middle panel).For each cell type visualized, a merged image in association with DAPIstaining is shown in the right panel. A typical punctuate mitochondrialpattern was observed in cells expressing the recoiled ATP6 polypeptides,when the Flag antibody was used. This indicates that fusion ATP6proteins localized to mitochondria.

Immunocytochemistry to detect the flag epitope in HeLa cells transientlyor stably transfected with the four pCMV-Tag 4A vectors showed a typicalpunctuate mitochondrial pattern, suggesting that the fusion Atp6 proteinhad been localized within the mitochondria (FIG. 3). Indeed, thistypical punctuate mitochondrial pattern was also observed using eitherthe mitochondrion-specific dye Mito Tracker Red or specific antibodiesanti-ATP synthase subunit beta. HeLa cells transfected with the emptypCMV-Tag 4A vector were used as negative controls and showed a diffusecytoplasmic distribution but with a low intensity (FIG. 3). Thelocalization patterns of the different Atp6 peptides which synthesiswere directed by the four pCMV-Tag 4A vectors were essentially identicalconfirming that both COX10 and SOD2 sequences successfully allowed thereengineered Atp6 protein to localize to the mitochondria in vivo.

Translocation of the Fusion Atp6 Protein into Mitochondria of HeLa Cells

To determine whether reengineered ATP6 gene products are efficientlyimported into mitochondria in vivo, mitochondria isolated from stablytransfected HeLa cells were subjected to western blot analysis (FIG. 4).We visualized two forms with anti-flag antibodies of approximately 30and 20 kDa, representing the precursor and mature forms of the recodedATP6 protein.

The predicted molecular weights of both proteins are respectively 34 and30 kDa, larger than the ones implied by the molecular weight markers.This discrepancy has often been observed when extremely hydrophobicproteins were migrated in SDS-PAGE. In general, the electrophoreticmobility on SDS-PAGE of proteins encoded by mtDNA is higher than the oneexpected for their theoretical molecular weights.

The steady-state levels of both polypeptides are similar in the two celllines examined: cells transfected with MTS COX10-nATP6 vector (MTSCOX10-nATP6), and cells transfected with MTS COX10-nATP6-COX10 3′UTR(MTS COX10-nATP6-3′UTR).

To determine the amounts of the recoded ATP6 polypeptides produced inHeLa cells expressing either SOD2^(MTS) ATP6-3′UTR^(SV40) or SOD2^(MTS)ATP6-3′UTR′^(SOD2) vectors, we compared six independent mitochondrialextractions (FIG. 14A). Both precursor and mature polypeptides wereequally abundant in mitochondria from each cell line, indeed theexpression of SOD2^(MTS) ATP6-3′YTR^(SV40) vector leads to anaccumulation of 61.4%±6 of the precursor form. Instead, SOD2^(MTS)ATP6-3′UTR^(SOD2) vector directed the synthesis of 64.4%±6.5 of theprecursor. These values were not significantly different according tothe paired Student's t-test. Similar results were obtained when totalextracts from each cell line were examined by Western blotting. Thisdata is in agreement with the overall amounts of hybrid ATP6 mRNAsdetected when total RNAs from cell lines expressing either SOD2^(MTS)ATP6-3′UTR^(SV40) or SOD2^(MTS) ATP6-3′UTR^(SOD2) vectors were subjectedto RT-PCR analyses (FIG. 12A). Therefore, the steady-state levels of theATP6 precursor and its ability to recognize the TOM complex in the outermitochondrial membrane do not depend on the presence of the SOD2 3′UTR.Notably, the relative proportions of ATP6 precursor and mature formswere analogous to the ones shown in cells for highly hydrophobicproteins en route to the mitochondria.

When mitochondria were treated with 150 or 200 microgrammes/ml ofproteinase K (PK) the precursor forms of the fusion ATP6 protein weresensitive to proteolysis in both cells lines. In contrast the matureform of ATP6 is resistant to PK digestion, especially in cellsexpressing the MTS COX10-nATP6-COX10 3′UTR transcript. Indeed, in thesecells the amount of the mature ATP6 protein is approximately 185% higherthan in cells expressing the MTS COX10-nATP6 mRNA. These data stronglyindicates that not only the precursor polypeptide is correctly addressedto the surface of mitochondria, as we observed by indirectimmunofluorescence (FIG. 3), but also that it was efficientlytranslocated into the organelle and correctly processed. Moreover, FIG.4 shows that the quantity of the mature form of the Atp6 protein and the65 kDa ATPalpha protein inside the mitochondria were quite similar afterproteinase K digestion. Therefore, the use of COX10 MTS allows anefficient mitochondrial translocation of the recoded ATP6 protein, andwhen COX10 MTS is combined to the 3′UTR of COX10 a significant moreefficient in vivo translation/import of the allotopically expressed ATP6gene is obtained.

FIG. 14B upper panel shows a schematic representation of thetheoretically expected ATP6 import intermediate. The hydrophobicpassenger ATP6 precursor can be trapped en route to the matrix and amitochondrial processing peptidase can cleave the MTS. Nevertheless, therest of the protein remained accessible to PK action and thereforebecoming undetectable on Western blotting. Instead, the fraction of theATP6 protein which can be completely translocated is insensitive toPK-induced proteolysis and can therefore be integrated into the innermitochondrial membrane, hence, remaining detectable on immunoblotting.

FIG. 14B shows that precursor forms of the fusion proteins weresensitive to proteolysis in both cell line examined. Nearly all the ATP6precursor signal disappeared after PK digestion, so precursors that wereengaged in the process of translocation or loosely attached to the outermitochondrial membrane but not fully translocated, were entirelydigested (FIG. 14B, middle panel). In contrast, a significant amount ofthe mature form of ATP6 is resistant to PK digestion, indicating itslocation inside the organelle. To examine the levels of another complexV protein in these cells, immunoblots were performed using anti-ATPsynthase α antibody. This naturally imported mitochondrial protein waspresent at similar extents in all cells tested. Only one band ofapproximately 65 kDa was visualized suggesting that either we wereunable to discriminate the precursor and mature forms of this proteinunder the electrophoretic conditions used or precursor polypeptides werevery rapidly and efficiently translocated. Additionally, no majordifferences of the ATP synthase α signals were detected after PKtreatment, confirming the integrity of the mitochondrial isolations(FIG. 14B, middle panel). To compare the import efficiency of therecoded ATP6 proteins in cells transfected with either SOD2^(MTS)ATP6-3′UTR^(SV40) or SOD2^(MTS) ATP6-3′UTR^(SOD2) vectors, we measuredthe amount of the mature form of ATP6 insensitive to PK digestion ineach cell line, after normalization with the amount of ATP synthase αresistant to PK proteolysis. Results for six independent mitochondrialextractions subjected to immunoblotting analyses were shown in FIG. 15B,lower panel. Overall results show that both SOD2 signals lead to a highefficient import of the recoded ATP6 precursor. Remarkably, the level ofthe mature form insensitive to PK proteolysis in cells transfected withSOD2^(MTS) ATP6-3′UTR^(SOD2) (1.28±0.24) was 1.8 fold higher than incells expressing the SOD2^(MTS) ATP6-3′UTR^(SV40) (0.71±0.12). Thedifference measured was significant according to the paired Student'st-test (P<0.0022, n=6). This observation could be related to the higherenrichment in the mitochondrion-bound polysomes of the correspondingmRNA (FIG. 12B).

The question arises whether imported reengineered ATP6 proteins wereassembled into the ATP synthase complex. The complex is organized inF0-F1 domains, F1 sector is a water-soluble unit located in the matrixand having the ability to hydrolyse ATP. The F0 domain is embedded inthe inner membrane and is composed by hydrophobic subunits forming aproton pathway. ATP6 is an intrinsic protein of F0, composed of fiveputative transmembranous α-helices. In contrast, ATP synthase α is alocated in the matrix F1 domain. Studies performed with bovine heartmitochondria demonstrated that ATP6 was degraded at a very low rate whenF0 subunits were subjected to trypsine treatment. Therefore, we treatedmitochondria with both PK and Triton X-100 (1%). The detergent disruptsboth mitochondrial membranes and theoretically leads to the entireproteolysis of mitochondrial proteins, demonstrating their localizationsomewhere inside the organelle in a protease-sensitive form. FIG. 3Cshows that indeed ATP synthase α was fully digested by PK; instead asignificant amount of ATP6 remained insensitive to PK proteolysis. Thisresult suggests that the recoded ATP6 was assembled into complex V.

Discussion:

Recent, epidemiological studies demonstrated that as a group, disordersof the mitochondrial function affect at least 1 in 5000 of thepopulation, making them among the most common genetically determineddisorders. In spite of the fact that over the last decade, theunderlying genetic bases of several mitochondrial diseases involvingcentral nervous system degeneration, no effective therapy is availablefor mitochondrial disorders. Pathogenic point mutations of genes encodedby the mitochondrial genome have been described as the cause of manymitochondrial disorders. A possible therapeutic approach is therefore toexploit the natural mitochondrial protein import pathway. The basicconcept is to introduce a wild-type copy of the mutated mitochondrialgene into the nucleus and import normal copies of the gene product intomitochondria from cytosol. This concept has been termed allotopicexpression and several reports in yeast described that a number of nonmitochondrial polypeptides can be relocated to the mitochondrial matrixsimply by conjugating a targeting sequence to their N-terminus. However,when this approach has been tried in mammalian cells using different MTSand genes encoded by mtDNA, precursors were not imported efficientlyinto mitochondria. By consequence, the rescue of mitochondrial defect inpatient cells was not only partial but also temporary [7]. Therefore, uptoday the spectrum of mtDNA encoded polypeptides than can besuccessfully expressed and integrated into mitochondrial respiratorychain complexes is very limited. This limitation is thought to be theconsequence of the high hydrophobicity nature of mtDNA encoded proteins,which possess transmembrane domains refractive to mitochondrial import.The precursor synthesized in the cytosol could lack the import-competentstructure required for an efficient mitochondrial membranetranslocation.

The concept of mesohydrophobicity is likely to be an important factorfor mitochondrial import competency. Mesohydrophobicity describes theaverage hydrophobicity in a window of 60-80 amino acids, together withthe calculation of the most hydrophobic 17-amino acid segment. Thiscalculation could predict importability of hydrophobic peptides. Usingtheir algorithm, we analyzed this correlation to assess themitochondrial importability of SOD2^(MTS) ATP6 gene product and comparedto ATP6, COX8 and SOD2 polypeptides as well as the previously testedfusion protein COX8^(MTS) ATP6: as the wild-type ATP6, both fusionproteins examined cannot be translocated into mitochondria, mainly dueto the high hydrophobicity of ATP6. Hence, a possibility that can allowthe import of a recoded ATP6 protein into the organelle is that theprecursor is engaged in a co-translational pathway of import. Thereby,the precursor would be maintained in a loosely folded nonaggregatedconformation required for translocation through the mitochondrial importapparatus. To overcome this limitation and try to develop a morelong-term and definitive rescue of mtDNA mutations by allotopicexpression leading to its application in gene therapy, we decided toconstruct nuclear versions of the mtDNA encoded ATP6 gene in which weappended the signals intended for forcing the hybrid mRNA to localize tothe mitochondrial surface. We have chosen COX10 and SOD2 genes, whichtranscripts are enriched in the mitochondrial surface.

We were able to demonstrate that the association to a recoded ATP6 geneof both the MTS and 3′UTR signals leading to a mRNA delivery to themitochondrial surface unambiguously improves the feasibility of theallotopic approach for mitochondrial genes. Indeed, not only we wereable to visualize the protein in the mitochondria by indirectimmunofluorescence, but most important the amounts of the processed Atp6polypeptide inside the organelle were quite similar to the naturallyimported ATPalpha protein. This result strongly indicates that therecoded Atp6 precursor was efficiently imported, the improvement we wereable to produce as compared to other recent reports [1], [2], [3], [4]is certainly due to the fact that the hybrid mRNA was addressed to themitochondrial surface, therefore enhancing the coupling betweentranslation and mitochondrial import processes.

Most interestingly, we obtained a gradual improvement, indeed the use ofeither COX10 or SOD2 MTS alone, gave a good result in whichapproximately 50% of the mature ATP6 protein is translocated inside themitochondria. When each MTS was combined to the corresponding 3′UTR, atleast 85% of the mature ATP6 protein is insensitive to proteinase Kdigestion indicating that almost all the protein synthesized in thecytoplasm is successfully translocated inside the mitochondria.

To accomplish allotopic expression, the localization of an mRNA to themitochondrial surface has never been tried before. In the allotopicapproaches reported, even though different MTS were appended to recodedmitochondrial genes, all the constructs examined contained at their 3′extremities the SV40 polyA signal that does not lead to any specificsubcellular localization of the transcript. Our data clearly demonstratethat the association to a recoded ATP6 gene of both the MTS and 3′UTRsignals of the SOD2 gene leads to a high efficient delivery of thehybrid mRNA to the mitochondrial surface. This improves unambiguouslythe feasibility of the allotopic approach for this mitochondrial gene.Indeed, not only were we able to visualize ATP6 protein in themitochondria by indirect immunofluorescence but definitely the amount ofthe processed ATP6 polypeptide inside the organelle was quite similar tothe naturally imported ATP synthase subunit α, a Complex V component,such as is ATP6. These data strongly indicate that recoded ATP6precursors were successfully imported. The improvement we obtainedcompared to a recent report, which measured 18.5% of the precursortranslocated, is certainly due to the localization of hybrid mRNAs tothe mitochondrial surface. This specific localization obviously enhancesthe coupling between translation and import processes, therefore,diminishing the block of the precursor during its translocation throughthe TOM and TIM complexes. It is worth mentioning that we obtained agradual improvement on mitochondrial import of the ATP6 precursor. Whenboth the MTS and the 3′UTR of SOD2 were combined, the amount of fullytranslocated ATP6 protein was 1.8-fold higher than when just the MTS waspresent. This is likely related to the improvement of mRNA sorting tothe mitochondrial surface when both cis-acting elements of SOD2 wereassociated to the recoded ATP6 gene. Remarkably, proteolysisinsensitivity of the translocated ATP6 protein in the presence of bothPK and Triton X-100 suggested that the protein could be correctlyassembled in the F0 domain of the respiratory chain Complex V. Notably,combining the cis-acting elements of the COX10 gene to the recoded ATP6gene, we obtained a very efficient mitochondrial import ability of thefusion protein. Indeed, COX10 mRNA codes for a highly hydrophobicprotein involved in Complex IV biogenesis, and SOD2 mRNA is enriched atthe mitochondrial surface in human cells.

We clearly demonstrated that the association to a recoded ATP6 gene ofboth the MTS and 3′UTR signals of either SOD2 or COX10 genes leads to ahigh efficient delivery of hybrid ATP6 mRNAs to the mitochondrialsurface, especially when both the MTS and the 3′UTR of SOD2 or COX10were associated to the reengineered ATP6 gene. This specific subcellularlocalization of hybrid mRNAs leads to a high efficiency in themitochondrial translocation of the recoded ATP6 proteins. Remarkably,when both the MTS and the 3′UTR of either SOD2 or COX10 were combined,the amount of fully translocated ATP6 protein was 1.8-fold higher thanwhen the MTS was associated to the cytosolic SV40 3′UTR. Therefore, theimprovement of mRNA sorting to the mitochondrial surface when bothcis-acting elements of SOD2 or COX10 were associated to the recoded ATP6gene definitely increase the amount of the processed ATP6 polypeptideinside the organelle which became quite similar to the naturallyimported ATP synthase subunit α, a Complex V component, such as is ATP6.Thus, by directing a hybrid mRNA to the mitochondrial surface wesignificantly improve the feasibility of the allotopic approach for theATP6 mitochondrial gene.

In conclusion, we optimize the allotopic expression approach for ATP6,by the use of mRNA targeting signals without any amino acid change inthe protein that could affect biologic activity.

This approach becomes henceforth available to rescue mitochondrialdeficiencies caused by mutations in mtDNA genes.

Example 2: Correct Mitochondrial Localization of the RecodedMitochondrial ND1 and ND4 Genes in Fibroblastes from LHON Patients

The three most common pathogenic mutations from LHON affect complex IND1, ND4 and ND6 genes with the double effect of lowering ATP synthesisand increasing oxidative stress chronically.

Since we have demonstrated that reengineered mitochondrial Atp6 proteinswere successfully translocated inside the mitochondria in HeLa cells(see example 1 above), we decided to synthesize recoded mitochondrialgenes ND1 and ND4. To ensure the efficient import of the allotopicallyexpressed proteins we appended to them signals which will direct thecorresponding mRNAs to the mitochondrial surface. We have chosen to usethe MTS of COX10 gene alone or in combination with its entire 3′UTR.FIGS. 5A and 5B illustrate the constructs obtained and the full-lengthsequences inserted in the pCMV-Tag 4A vector.

Material and Methods:

Cell culture and transfection: Fibroblasts were obtained from LHONpatients of the Hôpital Necker Enfants Malades, Paris, France(Département de Génétique). We cultured these cells with D-MEM mediumcomplemented with 10% of foetal bovine serum, pyruvate, gentamicin(0.01%), and 2 mM glutamine. When indicated cells were grown inglucose-free medium supplemented with 10 mM galactose.

Fibroblasts were transfected with EUGENE 6 transfection reagent asrecommended by the manufacturer (Roche Biochemicals, Indianapolis).Briefly, monolayer fibroblast cells were seeded a day beforetransfection at 50% confluence, so the next day they will be atapproximately 80% confluence, the cells were plated in a medium withoutantibiotics. 2 microgrammes of different plasmids purified with Quiagenplasmid midi kit (Quiagen; Valencia, Calif.) were used. Between 48 to 60hr later, 80% of the transfected cells were used for immunochemistryanalyses. The remaining 20% of cells were selected for neomycine, G418,resistance (selectable marker present in the pCMV-Tag 4A vector) at afinal concentration of 0.25 mg/ml. Stable clones were expanded forseveral weeks.

Optimized Recoding into Human Genetic Code

mtDNA has been recoded according to human genetic code, taking intoaccount the preferred codon usage in human:

TABLE 3 preferred codon usage in human Human preferred Source codonusage ARG CGA — CGC — CGG — CGU — AGA — AGG AGG LEU CUA — CUC — CUG CUGCUU — UUA — UUG — SER UCA — UCC UCC UCG — UCU — AGC — AGU — THR ACA —ACC ACC ACG — ACU — PRO CCA — CCC CCC CCG — CCU — ALA GCA — GCC GCC GCG— GCU — GLY GGA — GGC GGC GGG — GGU — VAL GUA — GUC — GUG GUG GUU — LYSAAA — AAG AAG ASN AAC AAC AAU — GLN CAA — CAG CAG HIS CAC CAC CAU — GLUGAA — GAG GAG ASP GAC GAC GAU — TYR UAC UAC UAU — CYS UGC UGC UGU — PHEUUC UUC UUU — ILE AUA — AUC AUC AUU — MET AUG AUG TRP UGG UGG % GC 63/42

ND1 and ND4 Constructs:

COX10 MTS-nND1-SV40 3′ UTR, COX10 MTS-nND4-SV40 3′ UTR, COX10MTS-nND1-COX10 3′UTR, and COX10 MTS-nND4-COX10 3′UTR were produced asdescribed above in example 1 for ATP6. The resulting sequences are shownon FIG. 5B (SEQ ID NO:23, 24, 25, 26, respectively).

Results:

Detection of ND1 Ailotopic Expression in Fibroblasts from LHON PatientsPresenting the G3460A ND1 Mutation

We analyzed the ability of the reengineered ND1 product to localize tomitochondria in viva. Immunocytochemistry analyses were performed todetect the flag epitope in fibroblasts, from a patient presenting theND1 gene mutated, transiently transfected with either COX10MTS-nND1-SV40 3′ UTR or COX10 MTS-nND1-COX10 3′UTR showed a typicalpunctuate mitochondrial pattern, suggesting that the fusion Nd1 proteinhad been localized within the mitochondria (FIG. 6). Indeed, thistypical punctuate mitochondrial pattern was also observed using specificantibodies anti-ATP synthase subunit beta. Cells transfected with theempty pCMV-Tag 4A vector were used as negative controls and showed adiffuse cytoplasmic distribution but with a low intensity (FIG. 6). Thelocalization patterns of Nd1 peptides which synthesis were directed bythe two vectors examined were essentially identical confirming thatCOX10 sequences successfully allowed the reengineered Nd1 protein to beaddressed inside the mitochondria.

Detection of ND4 Allotopic Expression in Fibroblasts from LHON PatientsPresenting the G11778A ND4 Mutation

Two plasmids directing the synthesis in the cytosol of a recodedwild-type ND4 gene were obtained. One of them, COX10 MTS-nND4-SV40 3′UTR, possesses appended to the N-terminus of the protein the sequencecorresponding to the first 28 amino acids of Cox10. The second one,COX10 MTS-nND4-COX10 3′UTR, has in addition at the end of the ORF thefull-length 3′UTR of COX10. Fibroblasts from a patient presenting 100%of mtDNA molecules with the G11778A ND4 mutation were transientlytransfected with either one of these plasmids. 60 h later cells werefixed and visualized to determine the ability of the COX10 sequences totarget the recoded protein to the mitochondria. FIG. 7 shows that inboth cases the fusion MTS Cox10ND4Flag protein did have a punctuatestaining pattern, which is very similar to the one observed for the samecells with the naturally imported mitochondrial protein ATP synthasesubunit beta. Thus, implying that the recoded Nd4 fusion protein wasimported into mitochondria.

In conclusion, as for the mitochondrial ATP6 gene, we were able tooptimize the allotopic expression approach for ND1 and ND4 genes, by thesimply use of mRNA targeting signals without any amino acid change inthe protein that could affect biologic activity.

Growth Ability of LHON Fibroblasts in Galactose Medium

Fibroblasts presenting the G3460A ND1 mutation were grown withgalactose, which slowly enters glycolysis as compared to glucose. FIG. 8shows major differences in cell growth after six day culture:fibroblasts presented a severe growth defect, less that 10% of the cellssurvived in medium containing galactose as compared to cells seeded inglucose-rich medium. Stably transfected fibroblasts with the MTSCOX10-nND1-COX10 3′UTR vector had a markedly improved rate of growth ingalactose compared with that of non-transfected cells. This resultimplies that the mitochondrially imported recoded ND1 protein hadassembled successfully into functional complex I allowing, therefore, arescue of mitochondrial dysfunction in these cells.

Example 3: Rescue of Mitochondrial Deficiency Causing Human Diseases(Transfection of Fibroblasts from a NARP Patient)

We also determined whether the reengineered ATP6 protein would be ableto rescue mitochondrial deficiency in cells having a mutated ATP6 gene.

We obtained fibroblasts from a patient presenting NARP disease caused bythe T8993G mutation in the ATP6 gene.

Fibroblasts were cultured on media containing sodium pyruvate andrelatively high amounts of FBS, more particularly:

-   -   on a medium containing glucose (D-MEM with L-glutamine, 4500        mg/L D-glucose, 110 mg/L sodium pyruvate 2.5 mM, FBS 15%,        uridine 28 microM), or    -   on a medium, which does not contain glucose, but contain        galactose (liquid D-MEM (1×), with L-Glutamine without Glucose,        sodium pyruvate 2.5 mM, galactose 10 mM, FBS 15%, uridine 28        microM).

Stably transfected cells expressing the nuclear version of ATP6associated with either SOD2 MTS alone, or in combination with SOD23′UTR, were obtained. Respiratory chain activity has been examined bythe ability of these cells to grow in a medium in which glucose has beenreplaced by galactose for either 10 or 20 days. NARP cells expressingthe empty vector had a low survival rate (30%). Cells expressing ATP6with either the MTS of SOD2 or both the MTS and the 3′UTR of SOD2present a growth survival of approximately 60%. If the selection wasmaintained for 20 days, the survival growth rate of cells expressing ouroptimized vectors was superior to 80% (FIG. 16). Only subtle differencesof survival rate were observed for fibroblasts expressing either thevector with both the MTS and 3′UTR of the SOD2 gene or the vector withthe SOD2 MTS associated to the SV40 3′UTR. This, is certainly due to thefact that these cells are heteroplasmic for the T8993G mutation, indeedthey possess approximately 10% of the wild-type gene. Therefore, intheir mitochondria probably 10% of a functional ATP6 protein could beassembled in Complex V. We can envision that the expression of thevector with SOD2 MTS associated to the SV40 3′UTR, will lead to themitochondrial import of enough ATP6 protein to allow the cells to growtha good rate in galactose medium.

Preliminary measures of the real amount of ATP produced in vitro byfibroblasts expressing either ones of our vectors clearly show adifference in the activity of Complex V related to the presence ofeither SOD2 3′UTR or SV40 3′UTR. Hence, when compared to controlfibroblasts (100% of ATP synthesis in galactose medium) NARP fibroblastsexpressing the vector with SOD2 MTS associated to the SV40 3′UTR had50%, representing an increase compared to non transfected NARP cells(30%) but was less important when compared to the amount found in cellsexpressing the vector which combines to the recoded ATP6 gene both theMTS and the 3′UTR of the SOD2 gene (approximately 85%); cf. FIG. 19. Byconsequence, a more complete and efficient rescue of mitochondrialdysfunction is obtained when allotopic approach implies the presence ofboth the MTS and 3′UTR targeting signals.

TABLE 11 Rate of ATP Survival rate on synthesis on Fibroblasts galactosegalactose Control 100%  100%  NARP (mutated ATP6) 30% 30% NARP +cytosolic 3′UTR 60% 50% (SV40 3′UTR) NARP + mitochondrial 60% 85% 3′UTR(SOD2 3′UTR)

Example 4: Rescue of Mitochondrial Deficiency Causing Human Diseases(Transfection of Fibroblasts from LHON Patients)

The applicability potential of the improved allotopic expressionapproach of the inventors has been further confirmed by examining twoother mtDNA genes involved in LHON. The fibroblasts obtained presented atotal homoplasmy of the mutation; indeed all the molecules ofmitochondrial DNA are mutated.

Fibroblasts were cultured on media containing sodium pyruvate andrelatively high amounts of FBS, more particularly:

-   -   on a medium containing glucose (D-MEM with L-glutamine, 4500        mg/L D-glucose, 110 mg/L sodium pyruvate 2.5 mM, FBS 15%,        uridine 28 microM), or    -   on a medium, which does not contain glucose, but contain        galactose (liquid D-MEM (1λ), with L-Glutamine without Glucose,        sodium pyruvate 2.5 mM, galactose 10 mM, FBS 15%, uridine 28        microM).

The engineered nucleus-localized versions of ND1 and ND4 were obtained;ND1 and ND4 transcripts possess both at their 5′ and 3′ extremitiesCOX10 mRNA targeting sequences. Stable transfections of theseconstructions in fibroblasts from LHON's patients with either ND1 or ND4mutations were performed. Indirect immunofluorescence showed that bothproteins localize to the surface of mitochondria in vivo. The OXPHOSactivity of these cells has been also examined by growing in a galactoserich medium. Interestingly, fibroblast cells allotopically expressingthe wild-type ND4 protein showed a markedly improved rate of growth ongalactose medium. This improvement is higher when both the MTS and 3′UTRof COX10 were associated to the ND4 gene (54.3%) as compared to that ofmock transfected cells (8%) or to the cells transfected with the ND4gene associated to the MTS of COX10 and the cytosolic SV40 3′UTR (12.7%)(FIG. 17, MTS: ND4 associated to COX10 MTS and the SV40 3′UTR, 3′UTR:ND4 associated to both the MTS and 3′UTR of COX10). This data imply thatin spite of the presence in these cells of the ND4 mutated polypeptide,the wild-type protein was successfully imported into the organelle andassembled in Complex I. Preliminary experiments, of in vitromeasurements of ATP synthesis confirm these results, indeed inuntransfected cells very little ATP was synthesized in galactose medium(14% of the control level measured in healthy fibroblasts), when cellsexpress the ND4 gene associated to the MTS of COX10 and the cytosolicSV40 3′UTR an increased is observed (56%). Remarkably, this increase ismore important when cells express the ND4 gene associated to both theMTS and 3′UTR of COX10 (84%).

Hence, our results undeniably confirm that we have optimized theallotopic approach for three mtDNA encoded genes by the use of mRNAtargeting signals, without any amino acid change in the proteins. Thisis particularly the case when our vectors presented both the MTS and the3′UTR targeting signals of a mRNA which exclusively localized to themitochondrial surface.

TABLE 12 Rate of ATP Survival rate on synthesis on Fibroblasts galactosegalactose Control  100% 100%  LHON (mutated ND4)   8% 14% LHON +cytosolic 3′UTR 12.7% 56% (SV40 3′UTR) LHON + mitochondrial 54.3% 84%3′UTR (COX10 3′UTR)

Example 5: Transduction of Retinal Ganglion Cells

The inventors obtained, by in vitro mutagenesis, reengineered ND1, ND4,ND6 and ATP6 genes, which possess the most common mutations found inLHON's and NARP's patients: G3460A, G11778A, T144840 and T8993Grespectively. Both wild-type and mutated genes have been integrated inthe p-AAV-IRES-hrGFP vector, which will allow the production ofinfectious recombinant human Adeno-Associated Virus Type 2 (AAV2)virions. For all constructions, each nuclear version of mtDNA genes isassociated to the two mRNA targeting sequences of the COX10 gene, whichallow the enrichment of corresponding mRNAs at the surface ofmitochondria. In accordance with the present invention, this will ensurethe efficient delivery of the polypeptides inside the organelle.

Retinal ganglion cells (RGC) represent the primary cellular target ofthe pathogenic process of LHON disease.

The inventors purified RGCs from adult rat retina, thereby obtainingenriched RGC populations, and maintained them in culture for two weeks.Mitochondria are distributed along actin filaments and they specificallyconcentrated at the extremities of neuron extensions. The inventorstransfected these cells with the mutated version of the ND1 gene.Preliminary results showed that the expression at high levels of themutated protein during 8 days leads to an abnormal distribution ofmitochondria along the neurite and cone extensions (FIG. 18).

BIBLIOGRAPHIC REFERENCES CITED IN THE EXAMPLES

-   1. Owen, R., et al., Recombinant Adeno-associated virus vector-based    gene transfer for defects in oxidative metabolism. Hum. Gene    Ther., 2000. 11: p. 2067-2078.-   2. Guy, J., et al., Rescue of a mitochondrial deficiency causing    Leber Hereditary Optic Neuropathy. Ann. Neurol., 2002. 52: p.    534-542.-   3. Manfredi, G., et al., Rescue of a Deficiency in ATP Synthesis by    Transfer of MTATP6, a mitochondrial DNA-encoded gene to the nucleus.    Nature Genet., 2002. 30: p. 394-399.-   4. Oca-Cossio, J., et al., Limitations of allotopic expression of    mitochondrial genes in mammalian cells. Genetics, 2003. 165: p.    707-720.-   5. Sylvestre, J., et al., The role of the 3′UTR in mRNA sorting to    the vicinity of mitochondria is conserved from yeast to human cells.    Md. Biol. Cell, 2003. 14: p. 3848-3856.-   6. Ginsberg, M. D., et al., PKA-dependent binding of mRNA to the    mitochondrial AKAP121 protein. J. Mol. Biol., 2003. 327(4): p.    885-897.-   7. Smith, P. M., et al., Strategies for treating disorders of the    mitochondrial genome. Biochem. Biophys. Acta, 2004. 1659: p.    232-239.-   8. Carelli et al., Progress in Retinal and Eye Research, 2004.    23: p. 53-89

1. An expression vector configured for the efficient and stable deliveryof a protein into the mitochondrion of a mammalian cell, the vectorcomprising: a mitochondrion-targeting nucleic acid sequence (MTS); anucleic acid sequence encoding said protein in accordance with auniversal genetic code (CDS); and a 3′UTR nucleic acid sequence, located3′ of said CDS, configured to target mRNA expressed by the expressionvector in the nucleus to the mitochondrion-bound polysomes of themammalian cell, wherein, said MTS consists of a nucleic acid sequenceencoding the peptide SEQ ID NO: 46, and said 3′UTR nucleic acid sequenceconsists of a conservative variant or fragment of the nucleic acidsequence SEQ ID NO: 47, said conservative variant or fragment stillallowing for the mitochondrial targeting of the mRNA.
 2. The expressionvector of claim 1, wherein the CDS nucleic acid sequence encodes theNADH-ubiquinone oxireductase subunit 4 protein (ND4).
 3. The expressionvector of claim 1, wherein the nucleic acid sequence encoding thepeptide SEQ ID NO: 46 is SEQ ID NO:
 30. 4. The expression vector ofclaim 2, wherein the CDS nucleic acid sequence encoding ND4 is SEQ IDNO:
 29. 5. The expression vector of claim 1, wherein said mammalian cellis a human cell.
 6. The expression vector of claim 1, wherein thenucleic acid sequence encoding said protein has been optimized withpreferred human codon usage.
 7. The expression vector of claim 2,wherein the nucleic acid sequence encoding the ND4 protein has beenoptimized with preferred human codon usage.
 8. The expression vector ofclaim 1, wherein the CDS nucleic acid sequence encodes a proteinselected from the group consisting of mitochondrially encoded ATPsynthase 6 (ATP6), mitochondrially encoded NADH dehydrogenase subunit 1(ND1), and NADH-ubiquinone oxidoreductase subunit 4 (ND4).
 9. Theexpression vector of claim 8, wherein the CDS nucleic acid sequenceencoding said protein has been optimized with preferred human codonusage.
 10. The expression vector of claim 8, wherein the CDS nucleicacid sequence is selected from the group consisting of SEQ ID NO: 29,SEQ ID NO: 28, and SEQ ID NO:
 27. 11. The expression vector of claim 1,wherein said 3′UTR nucleic acid sequence consists of the nucleic acidsequence SEQ ID NO:
 47. 12. The expression vector of claim 1, whereinsaid 3′UTR nucleic acid sequence consists of a conservative variant ofthe nucleic acid sequence SEQ ID NO: 47, said conservative variant stillallowing for the mitochondrial targeting of the mRNA.
 13. The expressionvector of claim 1, wherein said 3′UTR nucleic acid sequence consists ofa fragment of the nucleic acid sequence SEQ ID NO: 47, said fragmentstill allowing for the mitochondrial targeting of the mRNA.